Developmental neuroplasticity in a model of cerebral hemispherectomy and stroke

Neuroscience Vol. 95, No. 3, pp. 625–637, 2000 625 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00

Developmental neuroplasticity in hemispherectomy and stroke

Pergamon PII: S0306-4522(99)00482-0 www.elsevier.com/locate/neuroscience

COMMENTARY DEVELOPMENTAL NEUROPLASTICITY IN A MODEL OF CEREBRAL HEMISPHERECTOMY AND STROKE J. R. VILLABLANCA* and D. A. HOVDA† Departments of Psychiatry and Biobehavioral Sciences, and Neurobiology, Mental Retardation Research Center and Brain Research Institute, University of California, Los Angeles, CA 90024-1759, U.S.A.

Abstract—Cerebral hemispherectomy, a last resort treatment for childhood epilepsy, is a standard procedure which dramatically illustrates the resilience of the brain to extensive damage. If this operation, also mimicking long-term, extensive unilateral capsular stroke, is performed in postnatal cats of up to 60 days of age, there is a remarkable recovery/sparing of neurological functions that is not seen when the lesion occurs during late fetal life or in adulthood. A long-term effect at all ages is loss of neurons in bilateral brain areas remote from the resection site. This is pronounced in adult cats and shows intriguing, paradoxical features in fetal animals, but is substantially attenuated in neonatal cats. Similarly, large-scale reinnervation of subcortical sites (sprouting) by neurons of the remaining, intact hemisphere is prominent in young cats, but not in fetal or adult animals. These and other restorative processes (described herein) in young postnatal animals are matched by relatively higher rates of local cerebral glucose utilization, supporting the notion that they underlie the improved behavioral outcome. Thus, during a critical, defined stage of maturation, presumably common to higher mammals including humans, the brain entirely remodels itself in response to extensive but focal injury. Perhaps the molecular environment allowing for rescue of neurons and enhanced reinnervation at a specific developmental stage could be recreated in subjects with brain lesions at less favorable ages, thereby helping to restore circuitry and spare neurons. However, replacement via transplantation of neurons eliminated by the damage appears to be crucial in attempts to further preserve cells located remotely but yet destined to die or decrease in size. This article presents abundant evidence to show that there is a surprisingly comprehensive long-term morphological remodeling of the entire brain after extensive unilateral damage and that this occurs preferentially during a discrete period of early life. Additional evidence strongly suggests that the remodeling underlies the outstanding behavioral and functional recovery/sparing following early cerebral hemispherectomy. We argue that this period of reduced brain vulnerability to injury also exists in other higher mammals, including man, and suggest ways to enhance restorative processes after stroke/hemispherectomy occurring at other ages. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: neuroplasticity, developmental brain damage, cerebral hemispherectomy, stroke.

CONTENTS

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INTRODUCTION 1.1. Cerebral hemispherectomy HEMISPHERECTOMY IN EARLY POSTNATAL AND IN ADULT CATS 2.1. Behavioral outcome 2.2. Developmental reinnervation of partially denervated targets 2.3. Neuron loss and tissue atrophy 2.3.1. The thalamus and associated nuclei 2.3.2. The caudate nucleus 2.4. Cerebral metabolism THE FETAL BRAIN 3.1. Behavioral outcome 3.2. Paradoxical morphological changes 3.2.1. The thalamus and the neocortex. 3.2.2. The caudate nucleus 3.3. Reinnervation studies 3.4. Cerebral metabolism A CRITICAL MATURATIONAL PERIOD FOR POST-INJURY BRAIN RESTORATION 4.1. Timed postnatal hemineodecortications 4.2. A critical maturational period for cats, rats and monkeys CONCLUSIONS AND PERSPECTIVES 5.1. Postnatal studies 5.2. Prenatal studies

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*To whom correspondence should be addressed. Fax: 1 1-310-206-5060. E-mail address: [email protected] (J. R. Villablanca) †Present address: Department of Surgery (Neurosurgery), UCLA School of Medicine, Los Angeles, CA 90024-7039, U.S.A. Abbreviations: CMP, critical maturational period of decreased brain vulnerability to injury; E, embryonic or fetal day; LCMRglc, local cerebral metabolic rate of glucose utilization; P, postnatal day. 625

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Perspectives 5.3.1. Two main processes for postnatal brain repair 5.3.2. The riddle of the fetal brain 5.3.3. The critical maturational period ACKNOWLEDGEMENTS REFERENCES

1. INTRODUCTION

1.1. Cerebral hemispherectomy When an entire cerebral hemisphere is removed early postnatally in cats, the rest of the brain continues to grow and, in adulthood, the animals do not appear different from normal litter mates. Many years prior to this surprising observation, in our laboratory I (J.R.V.) was watching in awe as a neurosurgeon skillfully removed almost the entire left half of the brain of a right-handed young boy. The boy, like many other children today, suffered pharmacologically intractable epilepsy, a life-threatening condition which, at best, makes life miserable and leads to progressive brain deterioration, and at worst, simply kills. It was c. 1950, the dawn of cerebral hemispherectomy, a procedure developed by Dandy, 19 which later fell into disgrace due to complications. 72,81 Today, with the drawbacks under control, 1,24,76,81 hemispherectomy is a standard procedure in the U.S.A. (e.g., about 80 cases at UCLA between 1986 and 1996 76). Just as dramatic, albeit much more frequent, is a major cerebral stroke or “brain attack”. As in the heart, arteriosclerotic plaques may occlude small arteries supplying the brain internal capsule and vicinity, typically only on one side. Or, different from the heart, these vessels may break and bleed. Since the occlusion or hemorrhage is usually extensive, the destruction of nerve pathways and nuclei involved in movement control, somatic sensation and vision is so widespread that the entire hemisphere is practically obliterated, which is why the long-term consequences of hemispherectomy closely mimic those of extensive stroke. I was fortunate to follow the boy with hemispherectomy intermittently for about 10 years and to compare his progress to that of many adult stroke patients in the neurology ward. Ever since, I was intrigued by the differences in outcome, e.g., while language developed to some extent in the young patient and he improved enough as to become self-supporting, aphasia persisted in older stroke patients and the neurological recovery was limited. Above all, I was fascinated by how much insult the young brain could take and still keep a resemblance of normal function. Today, albeit still limited, there is more than just anecdotal data to support the conclusion that the human brain withstands damage early postnatally better than in adulthood. Perhaps the best evidence is from language studies. 68,90 The visual field opposite to the resection side is also preserved to a limited extent in young patients, 75 and studies of cognition support the notion that children with hemispherectomy are better off when surgery is performed prior to two to three years of age. 4 Back in the research laboratory, a clear parallel between the human and feline conditions emerged since, early on, we found that cats with hemispherectomy in adulthood also did not recover as well as kittens with the same resection. For us, then, cerebral hemispherectomy became the ideal model to study long-term brain plasticity following neocortical injury. Since many of our cats are studied for up to three to four

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years, this also mimics the chronic condition of human hemispherectomy and stroke. Results in postnatal cats and reports in monkeys and rats led us to extend the research into fetal life. What follows is a brief account of how we now understand the processes of brain healing after analysing for years postnatal cats sustaining hemispherectomy, as well as animals with a more restricted prenatal cortical removal at ages ranging from the last third of gestation and into adulthood.

2. HEMISPHERECTOMY IN EARLY POSTNATAL AND IN ADULT CATS

2.1. Behavioral outcome We performed hemispherectomy in 5- to 20-day-old kittens and in adult cats. The entire neocortex and caudate nucleus were removed, but the thalamus and ventral diencephalon were spared (e.g., Fig. 3A). After waiting for at least six months, we measured a wide spectrum of behaviors. Cohorts of five to seven animals were used for testing groups of related behaviors, and this reduced the number of repeated measures per animal groups. The early-lesioned cats performed significantly better in 23 of over 25 tests employed, such that upon casual inspection, they were indistinguishable from normal litter mates. Only some highlights of this work are summarized here (details are given in a series of six published papers 11,12,31,33,100,102). In some of the tests, adult-hemispherectomized cats showed gross or measurable impairments, while neonatalhemispherectomized animals showed little or no deficits. For example, during locomotion, the former cats but not the neonatal-hemispherectomized ones showed persistent turning to the side of the removal (95% of turns), weakness of the contralateral limbs, and splayed toes and limb adduction (paw-print analysis), while in the face there was weakness of the contralateral eyelids with a wider eye opening (and also with a wider pupil cross-sectional area 100). Similarly, for sensory tests: while contralaterally adult hemispherectomized cats showed very long latencies to withdraw the forepaw from water and a diminished response to tactile stimulation of the face, neonatal-hemispherectomized cats were almost normal. 100 In another group of tests, both ageat-lesion groups showed impairments, but these were much more marked in adult-hemispherectomized cats. For example, none of the cats recovered the contact component of the pawplacing reactions, but other components were almost intact in neonatal-hemispherectomized cats, such that they could traverse a narrow plank, while adult-hemispherectomized cats could not even stand upright in the plank. 100 Similarly, in an assessment of the visual field, 33 both groups showed a substantial contralateral hemi-field defect, but while in adulthemispherectomized cats this amounted to a complete hemianopsia, we found sparing of vision for the nasal hemi-field portion in neonatal-hemispherectomized cats (out to a 458 angle). In one test, a binocular depth perception measure, both groups of animals showed similar impairments, and in

Developmental neuroplasticity in hemispherectomy and stroke

another test, alignment of the binocular axis, neonatal-hemispherectomized animals showed a slight strabismus, while cats with hemispherectomy in adulthood did not. 31 Of further interest was a group of tests in which hemispherectomized cats showed performance biases. For example, in one test, cats were presented with a narrow Plexiglas box with food at the bottom, which was placed in a corner of a larger chamber in such a position that food could easily be reached only by using the paw contralateral to hemispherectomy (the paretic paw). None of the cats performed this task spontaneously. 11 However, while neonatal-hemispherectomized cats “learned” to use the contralateral paw in just one session following food deprivation and restraining of the ipsilateral paw, adult-hemispherectomized cats, under the same conditions, required a mean of 7.6 training sessions of over 1 h duration to switch usage to the paretic paw (the same restraint—upper non-affected limb—has proven useful to alleviate paresis in stroke patients 114). Similar performance asymmetries were detected in other tasks, including a T-maze task, 12 holeboard performance 12 and in batting at an object presented randomly in the visual field. 11 In all three tasks, the bias for a sided performance was more marked in the late, compared to early-lesioned cats, and reversing this bias was substantially easier in the latter animals. Overall, not only recovery/sparing of function was far greater for early hemispherectomized cats but, in addition, these cats exhibited much greater behavioral plasticity. All animals used in the experiments reviewed in this article were obtained from the Mental Retardation Research Center breeding colony at UCLA and were offsprings of cats raised in the colony or obtained (adults) from local pounds. All efforts were made to minimize animal suffering and to reduce the number of animals used. All procedures were approved by the UCLA Chancellor’s Committee for Animal Research. 2.2. Developmental reinnervation of partially denervated targets In cats, as in humans, control of movements is exerted by descending pathways projecting overwhelmingly to only one side of the neuraxis (called the “dominant innervation”). Using anterograde axon tracing techniques (see Ref. 104), we assessed whether this normal modality of projections had changed in cats with hemispherectomy at least 10 months in advance. Injections of tritiated leucine-proline were made in the remaining sensorimotor cortex for anterograde tracing of pathways. Injections sites and axon terminal fields were reconstructed using autoradiography-processed tissue. Under dark-field microscopy, predetermined sites in subcortical nuclei showing labeled particles were displayed on a monitor; the particles were digitized and counted using image analysis software, with background counts being subtracted. Multiple sites were used for measurements in each selected nucleus, e.g., for the red nucleus, 24 sites in three coronal planes were analysed bilaterally. Mean estimated values of silver grain densities per nucleus (site) and per coronal plane were calculated, expressed as number of particles per 1000 mm 2, and compared between animal groups. As illustrated in Fig. 1, in early-lesioned cats we found an extensive remodeling of pathways descending from the remaining sensorimotor cortex, and ending in the thalamus, 103 red nucleus, 104 nucleus of Darkschewitsch, 93 dorsal column nuclei of the brain stem (gracile and cuneate 26) and the

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cervical spinal cord. 26 In all these sites, estimates of density of terminals showed a remarkable bilateral, homotypical distribution in early hemispherectomized animals, indicating reinnervation of the partially denervated, non-dominant side of the neuraxis [heterotypical innervation may have occurred at the posterior thalamic site in neonatal-hemispherectomized cats, where a very high value was found (237% relative to control; Fig. 1, A8); this site corresponds to the ventral posterior medial nucleus, where very low counts were detected in normal brains; see Ref. 103]. Surprisingly, some reinnervation was also found in adult-hemispherectomized cats; however, this was substantial (33% of normal) and reached statistical significance only for the red nucleus (which plays a role in motor control). Further analysis in the red nucleus demonstrated that the topography of distribution, as well as the ultrastructure of novel terminals, were in all aspects similar to those present in normal cats. 22 We reported, in addition, expansion of terminal fields of axons originating in the deep cerebellar nuclei and ending in the contralateral, decorticated, red nucleus 71 and ventral thalamus. 60 Neurons in the remaining primary visual cortex were also examined following injections of a retrograde tracer (horseradish peroxidase) in the contralateral superior colliculus. 2 Novel innervation by decussating fibers from visual cortex neurons was seen in the colliculus and this was greater for the early-lesioned cats (35% of normal value). However, it was notable that a smaller reinnervation (7%) detected in adult-lesioned cats was also significant. In addition, crosssectional area measurements showed that the somata of the projecting neurons were larger than normal. We have reported a similar finding for neurons of the contralateral ventrobasal thalamus, 99 where cells with a soma area greater than 100 mm 2, increased in size by 18% and 15% in adult- and neonatal-lesioned cats, respectively, and also in the ipsilateral substantia nigra 53 (see Section 3.3). To sum up, the remodeling of axon terminals following hemispherectomy had three important features. (1) There was large-scale sprouting in that it encompassed multiple levels of the longest motor tract (corticospinal), as well as cerebello-rubro-thalamic and corticotectal projection systems. (2) The novel innervation was, by far, more extensive in early-lesioned animals; however, and importantly, it was also present to a limited extent in adult-hemispherectomized cats. (3) These age-dependent morphological differences matched the behavioral outcome since, as mentioned, the sensorimotor as well as visual field functional recovery were greater in early-lesioned cats. To our knowledge this is the most comprehensive, large-scale reinnervation of targets ever reported in a single animal model. Post-lesion sprouting has been repeatedly documented, but only for single brain structures in the same animal. 64,65,67,82,92,96 Large-scale sprouting has also been reported in the neocortex of monkeys. 23 A debate lingers regarding whether the “reinnervation” fibers are new axon terminals, i.e. true reinnervation, or just persisting terminals which were present early in life and would have retracted if a lesion had not occurred. There are data supporting the latter view for corticorubral terminals in cats but not rats. 64 However, for the corticotectal pathway there is no evidence to support the presence of transient projections. 2,54 The strongest support for new innervation actually comes from our results in adult-lesioned animals, i.e. a significant corticorubral 104 and corticotectal 2

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Fig. 1. The reinnervation of subcortical targets partially denervated by hemispherectomy (red bars) by axons descending from the motor cortex of the intact hemisphere (purple lines) in neonatal hemispherectomized cats (central panel). Some reinnervation also occurred following hemispherectomy in adulthood (right panel). Estimated density of right motor cortex axon terminals in the thalamus, red nucleus (n.), dorsal column nuclei and cervical spinal cord of normal and left cerebral hemispherectomized (HEMI) cats are shown as percentage of the innervation in intact cats. Tritiated amino acids were injected into the right pericruciate (motor) cortex and computer-assisted counts of labeled particles were performed in preselected sites of each nucleus. The calibration bar indicates the estimated number of labeled particles per 1000 mm 2. The purple line represents the corticofugal pathway descending from the motor cortex. The blue bars represent values for the normal dominant innervation (i.e. largest in normal animals) which, for comparisons, were given a score of 100%. All other values are shown as percentage of the normal innervation. Values for the non-dominant innervation (opposite to blue sites) are represented by red bars. These illustrate the key result that, while the non-dominant innervation along the brain main descending motor pathway is typically minimal in normal animals, it increased substantially in cats with neonatal hemispherectomy in all nuclei measured, suggesting reinnervation of these lesion-denervated targets. In adult-hemisperectomized cats, there was a tendency to reinnervation in some sites, which reached significance for the red nucleus. Statistics are not shown because the values represent combined results for a number of samples in each nucleus. The original values (in Refs 26, 103 and 104) indicated significant increases relative to normal for all non-dominant sites of neonatal-hemispherectomized cats (except the nucleus cuneatus). A 10, A 9.5 and A 8.0 indicate coronal stereotaxic levels for thalamic measurements.

reinnervation, since normal adult cats exhibit minimal nondominant terminals (Fig. 1) and retraction of early projections is not an issue at this age. Thus, most likely, the reinnervating axons are new collaterals arising from normal unilaterally projecting neurons 64 or, as claimed, 67 they may originate from a separate set of neurons which redirect their axons after the lesion. Indeed, as we have proposed, 13 the specific mechanism that actually operates may depend on the brain maturational state at the time of lesion.

Abundant evidence from our studies and the literature supports the notion that post-lesion novel axonal projections do have a functional role, thereby contributing to behavioral recovery. 92 This is outlined briefly as follows. (1) The new synapses have normal ultrastructural profiles 22 and electrophysiological properties. 96 (2) They are generally homotypical and have a normal topographical distribution. 22,96,104 (3) Sectioning the novel projecting fibers eliminates their putative function 66,82,86 and, in addition, in our cats, as well as in

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stroke patients, removing the remaining hemisphere 101 or a second 62 stroke enhances the impairments created by the first hemispherectomy/stroke, respectively. (4) Electrical stimulation of the remaining motor cortex in animals, 43 or transcranial magnetic stimulation in hemispherectomy or stroke patients, 6,16 produces bilateral electromyographic responses or bilateral limb movements. (5) Studies of cerebral blood flow in stroke patients 15 and of regional glucose uptake in animals with unilateral brain lesions 33,87 (see Section 3.4) suggest that the newly formed synapses are functional.

these sites and the ipsilateral neocortex, such changes occurred trans-synaptically. An additional sign of degeneration in adult-hemispherectomized cats was that the packing density of presumptive glial cells in the ipsilateral thalamic ventrobasal complex increased by 86%, whereas there was no change in the neonatallesioned animals. 99 In contrast, contralaterally, there were decreases of 29% and 27% for adult- and neonatal-lesioned cats, respectively. A similar trend was seen in the other structures that we examined. 32,89,105

2.3. Neuron loss and tissue atrophy

2.3.2. The caudate nucleus. In order to assess whether selective anterograde degeneration also occurs after removing the neocortex, we chose the caudate nucleus for analysis, since this large nucleus receives, but does not send, direct projections to the cortex. The caudate was spared in some cats and since, compared to the thalamus, we expected more subtle effects, we used stereological methods of analysis. 52 In cats with hemineodecortication in adulthood, the volume of the ipsilateral caudate shrank by 18%, while the total number of neurons was reduced by 22%. In cats with decortication around postnatal day (P) 10, the volume did not change, even though there was an insignificant decrease in the total number of neurons (18%). Contralaterally, the caudate volume did not change. Therefore, although the neuronal loss for adult-hemispherectomized cats was about one-third as much as that seen for the thalamus, there was a clear anterograde impact upon the caudate. For both age-at-lesion groups, there was only a trend for an ipsilateral increase in the total number of glial elements. A loss of cortical inputs to the striatum occurs in neurological syndromes, including extensive stroke, cerebral palsy and Huntington’s disease. 94 In sum, neuron loss and tissue atrophy were the hallmark of the hemispherectomy studies. However, and overall, the animals lesioned early in life showed substantial sparing relative to cats injured in adulthood, and this was remarkable since, as for reinnervation, these results also matched the behavioral outcome for both groups. There are relatively few developmental studies of postlesion degeneration in animals. Most of them were conducted in rodents and, to assess unilateral effects, used the contralateral side as control. 8,27,45,56,78 The prevalent conclusion in rodents was that the younger the animal was at the time of brain damage the greater was the tissue loss (called the “Gudden Principle” 8,28). However, the present results sharply oppose that conclusion and this discrepancy will be explained below (see Section 4.1). In contrast, studies in cats, most of them in the visual system, support our age-at-lesion conclusions 63,75 (and see Ref. 89).

When nerve cells die, consequences are felt at least as far as axons of these neurons travel and/or as far as other neurons sending terminals to the site of destruction are located. In postnatal subjects, changes in remote loci are degenerative in nature and may range from shrinkage to death of the affected neurons. Other elements of the neuropile also react, particularly the glial cells. These processes have been known for some time, 8,27,56,105 but issues such as how they are influenced by development and how extensive they truly are have received little attention. In hemispherectomized cats, we examined a number of subcortical nuclei bilaterally, as well as the cerebral cortex of the remaining hemisphere (see Section 4.1). 2.3.1. The thalamus and associated nuclei. Most thalamic nuclei, and particularly the ventrobasal complex, have reciprocal connections with the neocortex. Therefore, the thalamus is markedly altered by extensive ipsilateral cortical damage. 56 What surprised us was the substantial impact of age-at-lesion, as well as the bilaterality of the effects. In adulthemispherectomized cats, the size of the ipsilateral and contralateral thalamus was reduced by 60% and 14%, respectively, while in neonatal-hemispherectomized animals there was no contralateral reduction and the ipsilateral atrophy was significantly lower (48%; based on cross-sectional area measurements at two coronal planes 99). For assessment of cellular changes (see Ref. 99), neuronal and glial cells were identified using standard morphological criteria but, in addition, cell bodies with cross-sectional area greater than 35 mm 2 were classified as neurons (and were divided into size categories for statistical analysis), while smaller elements were classified as glial and treated as presumptive glial cells (we might have over-estimated the number of glial cells by including very small neurons, albeit in a small proportion relative to the total glial cell population 35). In the ipsilateral ventrobasal nuclei, we found a large but age-linked neuron loss and atrophy, with the largest cells (cross-sectional area 100 mm 2 or greater) suffering the most. In adult-lesioned cats, cell packing density of the largest neurons was strikingly reduced by 82% relative to control and the soma size of those remaining shrank by 35%. This sharply contrasted with reductions of only 52% (density) and 22% (soma size) in neonatal-hemispherectomized cats. The dorsal lateral geniculate 89 and medial geniculate nuclei, 32 which are thalamic relay stations for visual and auditory pathways, respectively, showed a similar pattern of age-related degeneration. Furthermore, two structures not directly linked to the neocortex, the superior colliculus and the mammillary bodies, showed similar age-related changes, albeit less marked. 105 Since at least two synapses are interposed between

2.4. Cerebral metabolism Local cerebral metabolic rates for glucose utilization (LCMRglc) reflect synaptic transactions and are generally used to represent brain activity. 58,91 As measured with positron emission tomography in man using [ 18F]fluorodeoxyglucose or with autoradiography in small animals using [ 14C]2deoxyglucose, the rates allow functional imaging and correlations with behavior. 24 We measured LCMRglc in 50 sites bilaterally, including most brain structures, in long-term hemidecorticated cats. 34 There was a bilateral depression of the mean rate (in mmol/100 g/min), but this was substantially greater for cats hemidecorticated in adulthood, compared to

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neonatally (see fig. 1 in Ref. 34). Thus, collapsing across all brain structures, in adult-lesioned cats the mean decrease was of 34% and 31%, while in the P10 group it was of only 17% and 26%, for structures ipsilateral and contralateral to the resection, respectively. Of further interest was that ipsilateral sites with more neuronal loss and with reduced reinnervation, such as the ventrobasal thalamus of adult-lesioned cats, 99,103 were the most depressed, e.g., a reduction of 57% for the ventral posterior lateral nucleus and of 49% for the dorsal lateral geniculate nucleus, compared to only 35% and 19%, respectively, for cats with the neonatal lesion. In contrast, sites with little cell loss and substantial reinnervation, such as the ipsilateral red nucleus of neonatal-lesioned animals, 104 suffered the least, i.e. a reduction of only 9% compared to 23% for adult-lesioned cats. Therefore, this metabolic map reflected the nature and degree of the structural changes, and also predicted the animals’ behavioral recovery. 3. THE FETAL BRAIN

Regardless of inter-species discrepancies (see below), the “Early Lesion Effect”, also known as the “Kennard Principle”, 44,95 is still widely accepted, 8,68,74,95 i.e. the earlier the brain lesion occurs relative to birth the better the long-term outcome should be. Our findings in postnatal cats perfectly matched the “Principle”. However, does this mean that the decreased vulnerability to injury seen in neonatal animals would persist or be enhanced if damage occurred at an even earlier age? Fetal brain lesion work comparable to ours is scarce. In one report supporting that possibility, 25 monkeys with resection of the dorsolateral prefrontal cortex at the end of the second one-third of gestation showed remarkable behavioral sparing, as well as preservation of thalamic morphology, compared to animals sustaining a similar lesion but around P50. 3.1. Behavioral outcome Our efforts to produce viable fetal-hemispherectomized animals did not succeed. Kittens with removal between fetal (embryonic, E) days E43–E55 (birth is at E63–E65) grew some after the lesion, but they were all stillborn. 106 A discrete unilateral resection including the sensorimotor cortex (Fig. 2A, B) did yield subjects for study (albeit with a loss of about one-third of the animals). As described in Ref. 106, a smaller removal included the anterior, lateral and posterior sigmoid gyri (Broadman areas 4, 6 and 3a), while a larger resection extended to the sulcus ansatus, dorsally, and added the coronal gyrus (area 3b) and upper aspect of gyrus proreus (area 8). Compared to animals sustaining a similar neocortical resection, but at about P10, adult fetal-lesioned cats performed significantly worse in 16 of over 20 behavioral tests from the same battery applied to the hemispherectomized animals. 106,107 The impairments were similar to those seen in hemispherectomized cats, albeit generally not as marked, and included motor, sensorimotor and even visual-related defects. Such neurological deterioration was proportional to the size of the lesion 106,107 and thus appeared to be unrelated to the surgical trauma to the mother. In contrast, cats with a similar but neonatal lesion were almost normal, except for minor defects of the contact component of the paw-placing reactions and a tendency to a body turning bias.

3.2. Paradoxical morphological changes 3.2.1. The thalamus and the neocortex. Upon visual inspection of these brains, we were impressed by the disruption of cortical sulci and gyri patterns that in normal cats are always typical. 106 This anomaly was profound in animals with the larger resection and co-existed with gross reduction in size of the damaged hemisphere (Fig. 2A). In measurements which followed, 51 we found ipsilateral volume reductions of 27% for the neocortex (Fig. 2C, E) and of 26% for the thalamus (Fig. 2E). In contrast, P10 cats exhibited volume reduction restricted to the thalamus and only by 14%. 41 Microscopic inspection of these areas, including the neocortex, showed no gross cytoarchitectural abnormalities. In the fetal group, bilateral measurements of packing density of neurons and presumptive glial cells and of neuron soma size in the thalamic ventrobasal complex and nucleus ventralis lateralis revealed no changes. Therefore, we concluded that a substantial ipsilateral decrease in thalamic and neocortical volumes, with essentially no change in cell packing densities, cannot but signify a proportional decrease in the total number of neuronal and glial cells. However, since there were no signs of neuropile degeneration, we could hardly speak of thalamic (and presumably cortical) atrophy (necrotic degeneration), and decided to call it a post-lesion hypotrophy. 3.2.2. The caudate nucleus. Within the context of these generally regressive effects, one structure, the caudate nucleus, was a striking exception. To our surprise, the volume of the ipsilateral caudate increased (Fig. 2C) by 15% 49 and tended to increase contralaterally (8%). In contrast, animals with a neonatal lesion exhibited an 8% ipsilateral caudate shrinkage. 52 For both age-at-lesion groups, the neuronal and glial cell packing density, measured in both island and matrix compartments, did not change substantially on either side. Therefore, applying the same reasoning as for the thalamus, we concluded that the cortical removal in fetal cats had exactly the opposite effect upon the caudate, i.e. an increase in the total number of cells which resulted in caudate nucleus hypertrophy. 3.3. Reinnervation studies These results were not as interesting as for hemispherectomized cats. In both age-at-lesion groups, there was a tendency to reinnervation of the red nucleus and of thalamic nuclei ipsilateral to the ablation, but not of the caudate nucleus. 13 The novel innervation reached significance only for the red nucleus of fetal-lesioned cats, but it was small, i.e. 13% of the dominant innervation (compared to about 85% for neonatalhemispherectomized cats). The poor results were likely due to the much smaller cortical removal in frontal-lesioned cats, e.g., projections from areas surrounding the removed cortex might have remained intact and might have provided additional ipsilateral reinnervation. 13 Regarding the caudate nucleus, in cats a substantial bilateral innervation is already in place, with the non-dominant side reaching over 60% of the dominant innervation. 3 3.4. Cerebral metabolism There were substantial declines of the LCMRglc for the cats with prenatal resection. There was a mean bilateral

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Fig. 2. The unique reaction of the fetal brain to a unilateral, restricted neocortical lesion: while the thalamus and neocortex shrunk (E), the caudate nucleus experienced an unusual hypertrophy, particularly ipsilateral to the resection (arrow in A). (A, B) The dorsal aspect of brains of adult cats which had a similar resection of the left frontal pole (arrows) during the 47th day of gestation (A) and early postnatally (B, nine days of age). Note a marked size decrease of the damaged hemisphere in the brain with prenatal resection, which contrasts with no visible shrinkage after postnatal removal. In addition, the topography of the cortical surface was entirely changed in fetal-lesioned brains due to marked bilateral distortion of sulcal and gyral patterns, while following postnatal resection the typical pattern of feline sulci and gyri was preserved. (C, D) Coronal sections at the level of the head of the caudate to illustrate the paradoxical hypertrophy (particularly at left, arrow) of this structure only in fetal-lesioned cats (C), while no substantial change in caudate size occurred after postnatal removal (D). (E, F) The overall shrinkage of the ipsilateral hemisphere in fetal-lesioned cats is largely accounted for by thalamic (arrow) and cortical size decreases (E), which were not present in the postnatal-lesioned brain (F).

decrease of 31% for each hemisphere as a whole, i.e. collapsing across the 50 brain sites measured, compared to nonsignificant 19% ipsilateral and 22% contralateral declines for neonatal-lesioned animals. 109 These reductions occurred regardless of the direction of volumetric shifts, i.e. the ipsilateral thalamus and caudate nucleus were equally affected in fetal-lesioned animals. Since there were no signs of degeneration in the neuropile of the thalamus and caudate nucleus, the decreased metabolic rates are hard to understand. Hypothetically, since fetal-lesioned cats showed a volume reduction/distortion of the neocortex, this perhaps resulted in a considerable drop in synaptic densities in all sites receiving cortical projections, thereby determining a decrease

in synaptic transactions and hence a decrease in glucose utilization. 4. A CRITICAL MATURATIONAL PERIOD FOR POST-INJURY BRAIN RESTORATION

The studies in prenatal cats demonstrated that there is a limit to brain immaturity as a helpful factor in post-injury restoration. Together with the postnatal hemispherectomy results, this finding suggested that there might be a discrete period in development during which brain vulnerability to destructive lesions is reduced. In cats, such a period must begin around birth, since our oldest fetal cats were E55 and

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Fig. 3. The bilateral atrophy (cell loss) of the thalamus and remaining neocortex after long-term hemispherectomy is attenuated in cats which were younger at lesion time (P10, P30 and P60), with a peak protective effect at 30 days, thereby supporting the notion of a critical maturational period for decreased brain vulnerability to injury. (A) A coronal section of a brain with removal of the entire left neocortex in adulthood, which illustrates the dramatic decrease in size (atrophy) of the left thalamus, as well as some shrinkage effect upon the remaining hemisphere. (B) The decrease in thalamic volume on the side of the resection was substantial (P , 0.01) in all age-at-lesion groups, but showed an age-dependent gradient: cats with the resection at P10, P30 and P60 showed significantly (P , 0.05) less atrophy than those with removal in adulthood. (C) Contralaterally, there was also thalamic atrophy, but this was less severe, reaching significance only for groups lesioned at age P60 or older. (D) The neocortex of the remaining hemisphere was also affected, but atrophy reached significance only for the two older groups. The P30 group tended to be affected the least overall, suggesting a peak for a putative critical maturational period of decreased brain vulnerability to injury. *P , 0.05, **P , 0.01, compared to controls; 1P , 0.05, compared to adult-lesioned cats.

our youngest postnatal animals were P3–P5 at resection time. However, the postnatal boundary was undetermined. 4.1. Timed postnatal hemineodecortications We have studied adult cats with hemineodecortication sustained at P10, P30, P60, P90, P120 and in adulthood, and compared these groups behaviorally, 102 as well as by measuring the volume of the thalami and of the remaining neocortex. 85 For 13 of the 16 behavioral tests applied, performance was significantly better for cats with the resection at P10 and P30, and for 10 items there was an abrupt increment in impairments in the transition between the P30 and P60 groups, with the level of impairments increasing at P90 and through the adult groups. 102 Similar results were obtained for morphometry. 85 As shown in Fig. 3, in adult-lesioned animals the ipsilateral (Fig. 3B) and contralateral (Fig. 3C) thalami suffered 56% and 14% volume reductions, respectively, relative to controls. In contrast, for cats lesioned at P10 and P30, there was an ipsilateral size reduction of only 45% and 44%, respectively (with no substantial atrophy in the opposite, intact side). In these animals, the neocortex of the remaining hemisphere also experience atrophy 85 after hemidecortication (Fig. 3D), together with subtle changes in the shape of the cortical

mantle. 84 There was a volume decrease of 18% in cats with removal in adulthood, with no atrophy for the P30 group and only a tendency to shrink for the P10 animals (10%). The atrophy was likely due to neuropile degeneration consecutive to both a major direct loss of corpus callosum fibers—which reciprocally connect the left and right neocortices—and to a minor indirect mechanism, i.e. the contralateral thalamic atrophy trans-synaptically 20 affected the neocortex. Thus, the greatest sparing from atrophy for both structures occurred for the P30 group, with the P10 and P60 groups exhibiting more sparing than the other groups (Fig. 3). Once again, in these animals [ 14C]2-deoxyglucose utilization rates were also correlated with the morphological and behavioral outcomes, 108 in that the overall rates decreased as the age of the cats at surgery increased. 4.2. A critical maturational period for cats, rats and monkeys With the timed hemidecortications, we bracketed a discrete period of decreased vulnerability to brain injury in cats which begins around birth and which ends between P30 and P60, tapering protractedly thereafter. Since this approximately 50-day period overlaps with discrete morphogenetic events occurring simultaneously in the brain of normal cats, we have called it the critical maturational period (CMP) for optimal post-injury brain and behavioral restoration.

Developmental neuroplasticity in hemispherectomy and stroke

Specifically, at birth, the cat’s forebrain has already undergone peak neurogenesis, although morphogenesis is still unfinished. 30,55,97,113 However, two other maturational processes dominate early postnatally: naturally occurring cell death (or apoptosis 21,98), which follows overproduction of neurons, and excess synaptogenesis, which in turn is followed or overlaps with pruning of synapses. 7,17,88,112,113 Development of dendritic fields is closely linked to these events. 115 While these brain “sculpturing” processes 38 appear to be most active between birth and P45–P60, they also end protractedly thereafter. 7,21,30,55,88 Brain lesion studies in monkeys and rats suggest that the CMP also exists in other mammals. In rhesus monkeys, the best outcome of neocortical damage occurs when the resection is performed by the end of the second one-third of gestation. 25 In rats, the best results are obtained postnatally with lesions starting at about P7 and through a few days thereafter. 45–47 Therefore, the CMP of monkeys appears to course entirely during fetal life, while in rats it is postnatal, and in cats it straddles birth. Initially, it is hard to understand why the CMP should occur with such different timing relative to birth in these species, but the issue becomes irrelevant when considering, as in the paragraph below, that monkeys are born with a more mature brain than rats and cats, while at birth the cat brain (see above) is more mature than that of rats. What is highly relevant, though, is that the timing of the putative CMP for monkeys and rats also appears to overlap with the same brain “sculpturing” period as in cats. Specifically, in monkeys apoptosis is protracted. For the visual system, in the lateral geniculate nucleus apoptosis starts by the middle one-third of gestation and ends by E103, 111 while for the visual cortex, the density of neurons decreases by 36% between birth and six months postnatally. 70 Regarding synaptogenesis and pruning, in the visual cortex there is rapid cell loss between E60 and 2.5 years postnatally, with further loss stopping by about four years of age. 10,69 In rats, apoptosis in the neocortex starts at about P2, peaks at P7 and practically ends by P30. 73 Peak connectivity for transcallosal axons occurs during the first postnatal week, with pruning continuing into the second week, 39,40 while geniculocortical projections follow a slightly earlier time-course. 9,42 Therefore, for monkeys “sculpturing” is mostly a prenatal event, while for rats it courses almost entirely postnatally. For the above reasons, we have proposed 85,107 that the period of ontogenesis during which the number of neurons, synapses and dendrites is being adjusted provides the adequate plasticity required for a long-term restorative response of the brain to focal damage, thereby underlying the CMP.

5. CONCLUSIONS AND PERSPECTIVES

5.1. Postnatal studies These experiments point to the following conclusions. The best behavioral outcome occurred after hemispherectomy in the early postnatal period. In all hemispherectomized cats there was extensive atrophy/neuronal loss in structures ipsilateral to the removal and to a lesser extent in contralateral sites; however, this loss was substantially smaller in neonatallesioned cats. Also, in all lesioned animals, neurons of the remaining neocortex reinnervate partially denervated subcortical targets. This process was pronounced after early

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postnatal lesion, but still occurred to a significant degree in some sites after resection in adulthood. Concomitantly, local cerebral glucose utilization rates were less reduced in the brain of early- compared to adult-hemispherectomized cats. For these reasons, we have proposed 34,85,107 that the adaptive morphological restoration which follows brain lesions early during cats’ postnatal life underlies the better functional results, as reflected by glucose utilization rates, 34 and that these changes are collectively responsible for the enhanced behavioral recovery/sparing of younger compared to older lesioned subjects. Regarding glial cells, presumptively astrocytes, although we did not use cytological markers to identify these elements, their increase in adult-hemispherectomized cats was so impressive and reliable that this finding was most likely significant. In contrast, following lesions in young animals, either fetal or postnatal, there was a reliable tendency to a decrease in glial-type cells. As has been suggested, 59,61 shifts in glial cell populations may play a role in tissue repair. Other changes in the postnatal-lesioned brains that we have discovered included expansion of thalamic 60 and rubral 71 axonal terminal fields of cerebellar nuclei, increase in soma size of reinnervating neurons, 2,52 and increase in dopamine (D2) receptor density in the caudate nucleus of neonatal-hemispherectomized cats. 48 The sum of these events, together with the above reinnervation, cell loss/sparing and glial cell changes, contributed to an extensive remodeling which reshaped the entire post-hemispherectomy brain, thereby revealing a large potential for long-term post-injury plasticity, particularly but not limited to younger brains, that has truly amazed us. It was important that neonatal animals with a smaller neocortical resection showed only limited changes, with all measures, compared to hemispherectomized cats. This clearly indicates that the extent of the challenge (amount of tissue resected) is also a key factor in determining the magnitude of the brain reaction to injury. Indeed, had we only used a small lesion, the bulk of the present results would not have been achieved. In addition, hemispherectomy has allowed us to model the long-term consequences of extensive unilateral internal capsule stroke. In this regard, we have quoted several papers reporting experimental results in stroke patients 6,15,16,62,114 which were quite similar to some of our findings in cats and this, we believe, contributes to validate the model. 5.2. Prenatal studies In fetal cats, none of the morphological changes were restorative in nature and, consequently, both local glucose utilization and behavioral recovery were hindered. Instead, these animals were appealing due to the uniqueness of the post-lesion morphological events. On the one hand, in our own postnatal studies (and in the literature), we have never seen the combination of volume and cell number decreases (thalamus) and increases (caudate) in different structures of the same lesioned brain. On the other hand, neither have we never seen a dramatic disruption of neocortical topography caused by a focal lesion. How might this have happened? As mentioned, at the time of cortical resection neurogenesis is intense in the cat brain, but cortical–subcortical connectivity is not yet established. 36 For the thalamus, a likely scenario is that since cortical targets were partially eliminated

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by the resection, thalamocortical neurons generated at the time may have suffered apoptosis. Simultaneously, neurogenesis may have been down-regulated in order to fit the diminished number of targets and inputs. The resection deprived the caudate of potential cortical inputs, and therefore an adaptive shrinkage should also have occurred. However, we found exactly the opposite. We propose that enhanced inputs from other sites and/or new targets for striatal neurons might have over-compensated for lesion-induced loss. So far, we have only found suggestive changes in the substantia nigra, 53 where the cross-sectional area of neuronal somata increased in size (by about 24%), thus potentially providing expanded targets and afferences for caudate neurons. However, we found no evidence for increased neuron-sustaining projections from the contralateral sensorimotor cortex, 13 or for a possible developmental migration of cells from the putamen into the caudate. 50 5.3. Perspectives 5.3.1. Two main processes for postnatal brain repair. Of the two key processes which appear to determine the longterm brain reaction to unilateral neocortical injury at any age, i.e. reinnervation and neuron loss/degeneration, reinnervation of partially denervated targets by the remaining neocortex has a diminished role after lesions during fetal life and in adulthood, but it is robust during the CMP. Since it is likely that the basic events involved in axonal sprouting, including axon guidance and axon cone growth, operate quite differently at different stages of ontogenesis, further knowledge of the cellular and molecular bases of such processes in animals should greatly help to understand reinnervation and its potential to restore damaged circuitry, as well as to protect partially denervated neurons. If the vigor of reinnervation in young brains could be replicated later in life, that would give hope for circuitry restoration in adulthood. The other key process is the age-dependent loss of neurons in gray matter areas remote to the site of injury. This is a fundamental event which has received relatively little attention. Our results suggest that after postnatal lesions the neurons affected form two categories, those which are entirely lost and those which survive, albeit smaller in size. 2,89,99 It is reasonable to assume that this category includes mainly neurons which send most axons to and receive most inputs from the neocortical cells eliminated by the resections, typically the thalamocortical projection neurons. 20 As mentioned, the mechanism of death for these elements is combined retrograde and anterograde axonal degeneration (necrosis). It is possible 25,99 that the main reason why less neurons die after neonatal injury is that at lesion time surviving cells have more sustaining connections (than in adulthood) with neurons other than those removed by the lesion. Regarding the category of neurons which only shrink but do not die, in our model these are most likely the cells, typically caudate and red nucleus neurons, which in the mature brain share afferent and/or efferent connectivity with cells eliminated by the ablation, as well as with intact neurons in remote sites. Some of these cells might marginally subsist for a period after the lesion (due to connections with remote neurons) and, eventually, be rescued via other restorative events. The one that we may suggest is reinnervation by sprouting. Since this is much stronger in neonatal cats, this would further explain why a smaller number of neurons is lost

with lesions at that age. In prenatal cats, only the first category of neurons appear to exist after the lesion (since we did not find shrunken neurons). This makes sense, i.e. since connectivity has not yet occurred, sprouting and pre-existing connections are not available as mechanisms for repair, thereby explaining the all-or-none nature of neuron loss in fetal cats. Recognizing the above two categories of at-risk neurons prompts a practical suggestion for therapy. It is fair to assert that neurons in the first category have indeed lost their “raison d’eˆtre”, since having been deprived of targets and inputs they would no longer have a physiological role to fulfill. This implies, we believe, that the only way to preserve such neurons would be to replace the cells eliminated by the damage, thereby giving the neurons at risk a chance of reinsertion into their normal functional circuitry. This would entail transplantation of neocortical tissue (e.g., Ref. 110). It is an entirely different scenario for the neurons which only shrink, since in that case any means of preserving their remaining pre-existing innervation, or more so, of enhancing any intrinsic potential reinnervation, should lead to enhanced restoration. 5.3.2. The riddle of the fetal brain. Overall, the thalamic hypotrophy and the caudate hypertrophy suggest that, during a certain stage of ontogenesis, the brain has the potential to react to accidental loss of tissue by decreasing or increasing the number of cells in remote structures, thereby adjusting their respective volumes. Might different brain structures hypertrophy asynchronously depending on the timing of the prenatal lesion? Would that have functional, perhaps pathological, implications? Perhaps our fetal lesion model may be useful to study the molecular genetics of a putative “switching” mechanism which allows the brain to modulate neurogenesis following developmental injury; this might lead to intervention tools to control neuron numbers during ontogenesis. Post-brain lesion hypertrophy of a brain structure has rarely been reported in the literature. 18,57 Although the responsible mechanism(s) remain(s) elusive, our finding is relevant to numerous studies reporting caudate nucleus volume changes in developmental psychiatric disorders, including obsessivecompulsive 83 and attention deficit hyperactivity 14 disorders, Tourette’s syndrome, 77 and schizophrenia. 41 Of further interest is that a prenatal onset of pathology is suspected for some of these diseases. 41,77 Thus, our fetal brain lesion paradigm may serve as a model to study the pathophysiology of these syndromes. 5.3.3. The critical maturational period. Regarding the CMP, the suggestion that it is linked to a period of brain ontogenesis which is shared by all higher mammal species has advantages. First, it circumvents the confusion created by using birth as the reference time-point for comparing animal species in terms of brain vulnerability to injury, as in the “Kennard Principle”. Second, it allows extrapolation of data 5,85,107 from well-studied species, like the cat, to other animals, and particularly to humans (as already done for rats; see Ref. 5). A corollary is that, since the duration of ontogenesis is proportional to the life span of the corresponding species (see Section 4.2), the CMP duration should be longer in primates versus cats versus rats (which agrees with the literature 25,45–47,85,107). Accurate CMP bracketing may allow for better timing to study its cellular and

Developmental neuroplasticity in hemispherectomy and stroke

molecular underpinnings in animal models, which in turn may provide clues to manipulate such processes for therapeutic purposes. Although maturational data are still incomplete for humans, apoptosis appears to occur prenatally, relatively early in the cerebellum (dentate nucleus, between E17 and E230; see Ref. 29) and later on in the neocortex, with neuron numbers peaking by embryonic weeks 28–32 and stabilizing by birth. 79 Synaptogenesis, at least in the neocortex, appears to course postnatally, with synapse numbers peaking between two months and two years postnatally (depending on the cortical region 36,37,80). Pruning is very protracted and appears to continue into adolescence. 36,37 Therefore, the “sculpturing” period is, predictably, much longer in humans than in the mammals discussed above, such that it is tempting to cautiously propose that a putative CMP in humans should start by the beginning of the last trimester

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of gestation and continue through two to three years postnatally, ending protractedly thereafter. A practical consideration is that procedures to treat developmental brain damage, including hemispherectomy for childhood epilepsy, may achieve greater success when performed during this time interval.

Acknowledgements—This work was supported by NIH grants NS 25780, HD 05958, HD 04612 and NS 15654. Contributions from the following postdoctoral scholars in Villablanca’s laboratory are greatly appreciated: D. P. Adelson, F. Benedetti, W. J. Burgess, P. CarlsonKuhta, F. Go´mez-Pinilla, D. A. Hovda, C. Infante, L. Loopuijt, G. P. McAllister, Ch. E. Olmstead, T. D. Schmanke, B.L. Shook, B. J. Sonnier and R. I. Sutton. We are grateful to Professors V. R. Edgerton (UCLA), D. M. Feeney (University of New Mexico) and P. McKinley (McGill University) for their constructive and helpful criticism of this manuscript.

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