In vivo detection of developing vessel occlusion in photothrombotic ischemic brain lesions in the rat by iron particle enhanced MRI.

The aim of our study was to visualize developing vessel occlusion in focal cerebral ischemia in vivo. Cortical photothrombosis (PT) was induced in rats, which in addition received superparamagnetic iron oxide (SPIO) particles intravenously. When SPIO particles were applied simultaneously during illumination of the brain for induction of PT, animals showed a markedly hypointense cortical lesion on T2-weighted (T2-w) magnetic-resonance images (MRI). At 3 h after PT, this hypointense area was surrounded by a small hyperintense rim. At 48 h after PT the hyperintense rim had further extended, whereas the hypointense lesion core did not change in size or signal. On histological sections areas of signal loss on T2-w MRI corresponded to local accumulation of iron particles, which were trapped within vessel thrombi. When SPIO particles were applied at 2 h after PT, the lesion appeared hyperintense on T2-w MRI, but was surrounded by a small hypointense rim indicating ongoing vessel occlusion at its outer margins. In contrast, delayed SPIO application at 24 h after completion of PT produced a merely hyperintense cortical lesion on T2-w MRI. Correspondingly, no iron deposits were seen on tissue sections. In conclusion, early SPIO-enhanced MRI provides a reliable in vivo tool to delineate areas of developing vessel occlusion in experimental cerebral ischemia and identifies vessel thrombosis as one mechanism of secondary infarct growth in the PT paradigm. This new imaging technique may aid to evaluate antithrombotic treatment strategies in the future.     

Repeat traumatic brain injury in the developing brain

The Center for Disease Control estimates that there are 1.7 million brain injuries in the US each year with 51% of these injuries occurring during periods of cerebral development. Among this population there is a growing population of individuals with repeat traumatic brain injury (RTBI). While the exact incidence is unknown, estimates range from 5.6 to 36% of the TBI population. This review summarizes the clinical problems/challenges and experimental research models that currently exist. It is intended to reveal the critical areas that need to be addressed so that age-relevant clinical management guidelines can be established to protect this population.     

Seasonal rewiring of the songbird brain: an in vivo MRI study

The song control system (SCS) of songbirds displays a remarkable plasticity in species where song output changes seasonally. The mechanisms underlying this plasticity are barely understood and research has primarily been focused on the song nuclei themselves, largely neglecting their interconnections and connections with other brain regions. We investigated seasonal changes in the entire brain, including the song nuclei and their connections, of nine male starlings (Sturnus vulgaris). At two times of the year, during the breeding (April) and nonbreeding (July) seasons, we measured in the same subjects cellular attributes of brain regions using in vivo high-resolution diffusion tensor imaging (DTI) at 7 T. An increased fractional anisotropy in the HVC–RA pathway that correlates with an increase in axonal density (and myelination) was found during the breeding season, confirming multiple previous histological reports. Other parts of the SCS, namely the occipitomesencephalic axonal pathway, which contains fiber tracts important for song production, showed increased fractional anisotropy due to myelination during the breeding season and the connection between HVC and Area X showed an increase in axonal connectivity. Beyond the SCS we discerned fractional anisotropy changes that correlate with myelination changes in the optic chiasm and axonal organization changes in an interhemispheric connection, the posterior commissure. These results demonstrate an unexpectedly broad plasticity in the connectivity of the avian brain that might be involved in preparing subjects for the competitive and demanding behavioral tasks that are associated with successful reproduction.     

Phenobarbital modifies seizure-related brain injury in the developing brain

To investigate the potential role of drug therapy in preventing or exacerbating seizure-related brain injury in the prepubescent brain, we administered kainic acid to rats at postnatal day 35. Therapy with daily phenobarbital was started directly before or 1 day after kainic acid was administered, and was continued through postnatal day 153. Rats receiving phenobarbital had therapeutic concentrations during most of the 24-hour dosing period, but also experienced supratherapeutic peak concentrations. The animals were subsequently tested using the water maze (a measure of visuospatial memory), open field (a measure of activity level), and handling tests (a measure of emotionality). The frequency of spontaneous recurrent seizures was monitored during and after phenobarbital therapy. Kainic acid resulted in status epilepticus on postnatal day 35 in all the rats that received it but those receiving phenobarbital first manifested a shorter and less severe status epilepticus as compared to the rats given kainic acid alone. Rats starting phenobarbital immediately before kainic acid was administered did not differ from control rats on behavioral testing and had no subsequent spontaneous recurrent seizures and no histological lesions. Rats receiving kainic acid alone performed significantly poorer than did control rats in the water maze, were more aggressive, had histological lesions, and manifested spontaneous recurrent seizures. As compared to the group treated only with kainic acid, rats receiving kainic acid followed by phenobarbital at postnatal days 36 to 153 manifested similar aggressiveness and histological lesions, similar frequency of spontaneous recurrent seizures after phenobarbital taper, and even greater disturbances in memory, learning, and activity level. These results demonstrate that kainic acid–related injury can be prevented by a medication working through inhibitory mechanisms; that structural and functional damage in the prepubescent brain can be prevented through strategically timed pharmacotherapy; and that treatment of spontaneous recurrent seizures alone with daily exposure to phenobarbital does not decrease, and may actually exacerbate, damage in the kainic acid model.     

Plasticity and injury in the developing brain

The child's brain is more malleable or plastic than that of adults and this accounts for the ability of children to learn new skills quickly or recovery from brain injuries. Several mechanisms contribute to this ability including overproduction and deletion of neurons and synapses, and activity-dependent stabilization of synapses. The molecular mechanisms for activity-dependent synaptic plasticity are being discovered and this is leading to a better understanding of the pathogenesis of several disorders including neurofibromatosis, tuberous sclerosis, Fragile X syndrome and Rett syndrome. Many of the same pathways involved in synaptic plasticity, such as glutamate-mediated excitation, can also mediate brain injury when the brain is exposed to stress or energy failure such as hypoxia-ischemia. Recent evidence indicates that cell death pathways activated by injury differ between males and females. This new information about the molecular pathways involved in brain plasticity and injury are leading to insights that will provide better therapies for pediatric neurological disorders.     

Manganese-enhanced 3D MRI of established and disrupted synaptic activity in the developing insect brain in vivo

The antennal lobe of the sphinx moth Manduca sexta serves as a model for the development of the olfactory system. Here, the establishment of the glomerular synaptic network formed by the olfactory receptor axons and antennal lobe neurons at pupal stage P12 was followed by transection of the right antenna and - within 24 h - by injection of MnCl2 into the hemolymph. In vivo 3D MRI at 100 and 60 microm isotropic resolution was then performed at P13 to P17. Whereas the left antennal lobe revealed a pronounced increase of the signal-to-noise ratio (SNR) reflecting normal synaptic activity, the observation of only a small SNR increase within the right antennal lobe indicated the disruption of pertinent activity after antennal transection. The accumulation of manganese in the intact antennal system became observable within 3 h and lasted for at least 2 days after injection. Intra-individual comparisons between the right and left side yielded a statistically significant differential SNR increase in the left antennal lobe. Because such an effect was not observed in younger animals studied at pupal stages P10/P11, the MRI findings confirm the development of functional synapses in the antennal lobe of Manduca sexta by P13.     

Angiogenesis in developing rat brain: an in vivo and in vitro study

Brain capillary proliferation in postnatal rats was measured in vivo by [3H]thymidine autoradiography. Maximal capillary proliferation occurred between 5 and 9 postnatal days, and was 40 times greater than in the adult. To test the hypothesis that soluble angiogenesis factors play a role in this developmental vascularization of brain, we prepared extracts from the brains of 6-day-old rats at the peak of proliferative activity, and from adults when it was lowest. We assayed them using an in vitro growth system measuring [3H]thymidine incorporation into cultured brain capillary endothelial cells. Extracts prepared from either 6-day or adult rats and containing 150 micrograms/ml protein caused more than a 4-fold stimulation of the endothelial cells, increasing to 8-fold at a concentration of 1500 micrograms/ml. The presence of growth-promoting activity in brain extracts from both adult and immature rats suggests that soluble angiogenesis factors may be present in the brain throughout life, but are unavailable for stimulation of in vivo capillary growth unless released or activated by an appropriate stimulus.     

Pharmacology of N-methyl-D-aspartate-induced brain injury in an in vivo perinatal rat model

Intrastriatal injection of the glutamate analogue N-methyl-D-aspartate (NMDA, 25 nmol) in postnatal day (PND) 7 rats provides a rapid, sensitive, and reproducible assay in which potential neuroprotective strategies against excitotoxic neuronal injury can be examined in vivo. Brain injury is quantified 5 days postinjection by comparison of the weights of the injected and contralateral cerebral hemispheres. Intraperitoneal injections (15 minutes post-NMDA) of competitive and noncompetitive NMDA receptor antagonists attenuated the severity of NMDA-induced brain injury. The rank order of neuroprotective potency of these antagonists was CGS-19755 greater than DOIPG greater than dextromethorphan greater than HA-966. Of these compounds only the competitive antagonist CGS-19755 provided complete neuroprotection. NMDA-mediated brain injury was also reduced by the specific sigma receptor ligands +PPP and haloperidol (35% reduction). In contrast, drugs that reduce presynaptic neurotransmitter release (adenosine) or enhance neuronal inhibition (baclofen) were not effective against NMDA toxicity. Although all five of the anticonvulsants tested limited NMDA-induced seizure activity, only carbamazepine reduced NMDA-mediated brain injury (36% reduction). These findings extend earlier observations that NMDA receptor antagonists can limit NMDA-induced toxicity in vivo and suggest that sigma receptors contribute to the pathophysiology of NMDA-mediated brain injury in vivo. Furthermore, NMDA-induced seizures and brain injury appear dissociable in this in vivo model. The results illustrate important practical limitations of neuroprotection in vivo vs. in vitro.     

In vivo ethanol decreases phosphorylated MAPK and p70S6 kinase in the developing rat brain

Exposure to ethanol during pregnancy is detrimental to fetal development, and individuals affected by the fetal alcohol syndrome present a number of CN system dysfunctions including microencephaly and mental retardation. Recently, it has been suggested that ethanol-induced inhibition of glial cell proliferation may be relevant in the causation of microencephaly. In this study, we measured the developmental changes of MAPK (ERKl/2) and p70S6 kinase, which are considered to play a prominent role in cell proliferation, and their phosphorylated proteins in rat brain, and examined the effects of in vivo ethanol administration. MAPK and phospho-MAPK increased gradually after birth, and reached adult levels on postnatal day 21. In contrast, levels of both p70S6 kinase and phospho-p70S6 kinase decreased after birth. Exposure to ethanol (2-6 g/kg, from postnatal day 4 to 7) had no effects on MAPK or p70S6 kinase levels, but caused a dose-dependent decrease of both phosphoproteins. These results suggest that phosphorylation of MAPK and p70S6 kinase may represent relevant targets for the developmental neurotoxicity of ethanol, and may be involved in microencephaly.     

In vivo phosphorylation of myelin basic proteins in developing mouse brain: evidence that phosphorylation is an early event in myelin formation

Myelin basic proteins (MBPs) can be detected in 5-day mouse brain, even though there is very little myelin present. In addition to the 4 MBP components (with molecular weights of 14, 17, 18.5 and 21.5 kilodaltons), that are commonly observed, other larger MBP-related polypeptides are also present. We have investigated the possibility of MBP phosphorylation at early stages of development in order to establish the point at which this modification occurs in the assembly of myelin. In vivo isotope studies show that in the 5-day mouse brain 14-, 17-, 18.5- and 21.5- kilodalton MBPs are phosphorylated and that they may be present in an immature form of myelin. Larger MBP-related proteins with a molecular weight of 35 and 42 kilodaltons are also phosphorylated but appear to be present mostly in nonmyelin structures or compartments.     

In vitro and in vivo effects of chlorpyrifos on glutathione peroxidase and catalase in developing rat brain

Preliminary findings of a study on the role of oxidative stress in the developmental neurotoxicity of chlorpyrifos (CPF) indicates that in vitro exposure to 1-100 microM CPF or 1-100 nM CPF-oxon had no effect on the activity of glutathione peroxidase (GSHpx) in brain homogenates from postnatal day (PN) 21 rats, or on the activity of purified GSHpx. A single high-dose acute injection of 45 mg/kg CPF to PN19 rats also did not significantly alter GSHpx activity at PN21, in spite of extensive (72%) brain acetylcholinesterase (AChE) inhibition. However, catalase activity was significantly reduced by 28%. PN21 pups exposed maternally to a lower effective dose of CPF throughout development (dams injected with 50 mg/kg every 3 days) also had normal GSHpx activity, but a 30% increase in H2O2-independent NADPH consumption. Brain catalase activity in these rats was significantly increased by 24%. These preliminary data suggest that specific GSHpx activity is not altered by in vitro or in vivo exposures to CPF-oxon or CPF, but catalase and an unknown H2O2-independent NADPH-consuming factor were affected differentially depending on the type and timing of exposure.     

Ethanol-induced caspase-3 activation in the in vivo developing mouse brain

Recently several methods have been described for triggering extensive apoptotic neurodegeneration in the developing in vivo mammalian brain. These methods include treatment with drugs that block NMDA glutamate receptors, drugs that promote GABA(A) neurotransmission, or treatment with ethanol, which has both NMDA antagonist and GABAmimetic properties. A single intoxication episode induced by any of these agents is sufficient to cause widespread neurodegeneration throughout many brain regions. The cell death process transpires rapidly from early to late stages within several hours. As the neurons die, they become TUNEL positive and show, by both light and electron microscopy, all of the classical morphological characteristics of apoptosis. In the present study, using immunocytochemical methods, we document that ethanol intoxication of 7-day-old infant mice causes a widespread pattern of caspase-3 activation corresponding to the pattern of apoptotic neurodegeneration that is occurring simultaneously.     

Sexual dimorphism in the inflammatory response to traumatic brain injury

The activation of resident microglial cells, alongside the infiltration of peripheral macrophages, are key neuroinflammatory responses to traumatic brain injury (TBI) that are directly associated with neuronal death. Sexual disparities in response to TBI have been previously reported; however it is unclear whether a sex difference exists in neuroinflammatory progression after TBI. We exposed male and female mice to moderate‐to‐severe controlled cortical impact injury and studied glial cell activation in the acute and chronic stages of TBI using immunofluorescence and in situ hybridization analysis. We found that the sex response was completely divergent up to 7 days postinjury. TBI caused a rapid and pronounced cortical microglia/macrophage activation in male mice with a prominent activated phenotype that produced both pro‐ (IL‐1β and TNFα) and anti‐inflammatory (Arg1 and TGFβ) cytokines with a single‐phase, sustained peak from 1 to 7 days. In contrast, TBI caused a less robust microglia/macrophage phenotype in females with biphasic pro‐inflammatory response peaks at 4 h and 7 days, and a delayed anti‐inflammatory mRNA peak at 30 days. We further report that female mice were protected against acute cell loss after TBI, with male mice demonstrating enhanced astrogliosis, neuronal death, and increased lesion volume through 7 days post‐TBI. Collectively, these findings indicate that TBI leads to a more aggressive neuroinflammatory profile in male compared with female mice during the acute and subacute phases postinjury. Understanding how sex affects the course of neuroinflammation following brain injury is a vital step toward developing personalized and effective treatments for TBI.     

Localized in vivo H-MRS of traumatic brain injury

¹H-MRS examinations were carried out on 14 patients, recovering from traumatic brain injury (TBI), who were in a stabilized clinical status and showed a good clinical outcome. Magnetic resonance spectra were recorded in subcortical (SC) and mid-brain (MB) areas where no detectable lesions appeared under magnetic resonance imaging. These two brain areas were selected because they are crucial sites of damage due to the physiopathologic mechanisms of TBI. A significant increase in inositol and choline peaks was found in MB compared to a control group of healthy individuals, whereas lower N-acetyl-aspartate peaks in the same area were detected. Reduced levels in the latter metabolite were also evident in the SC area. A significant correlation emerged between the inositol concentration in MB and the Glasgow Coma Scale Score measured just after the trauma. No correlation was found between the Glasgow Outcome Scale (GOS) at the time of the ¹H-MRS examination and the peaks of all the metabolites. Our study demonstrated that ¹H-MRS is a sensitive tool to evidentiate brain metabolic damage after TBI even in areas with lesions that are not detectable with current imaging techniques. The present research also shows an association between the alteration in one of the brain metabolites and the clinical parameters of TBI severity, but does not provide a clinical index of the patient's recovery. Further longitudinal studies on more conspicuous groups of patients with TBI could help to clarify whether metabolite modifications revealed by ¹H-MRS could be predictive of clinical outcome.     

Pediatric stroke: rehabilitation of focal injury in the developing brain

This review provides an overview of pediatric ischemic stroke to serve as a foundation for the discussion of rehabilitation strategies following focal injury in the developing brain. Cerebrovascular disease is an important cause of acquired brain injury in neonates and children. Ischemic strokes are caused by a multitude of risk factors and advances in neuroimaging have improved diagnosis and understanding of pathophysiology. Pediatric stroke provides the ideal model for the study of injury and recovery in a plastic nervous system. Though their brains likely posses greater potential and unique reorganizational skills, most children suffer neurological morbidity after stroke. An improved understanding of these systems is helping us understand, validate, and improve traditional approaches to rehabilitation while opening the door to new opportunities to improve outcome. All aspects of a patient's function, from the physical to psychological, including issues unique to children and their families, must be addressed and are reviewed. New advances and future directions for research are highlighted.     

Neuropathological and biochemical features of traumatic injury in the developing brain

Trauma to the developing brain constitutes a poorly explored field. Some recent studies attempting to model and study pediatric head trauma, the leading cause of death and disability in the pediatric population, revealed interesting aspects and potential targets for future research. Trauma triggers both excitotoxic and apoptotic neurodegeneration in the developing rat brain. Excitotoxic neurodegeneration develops and subsides rapidly (within hours) whereas apoptotic cell death occurs in a delayed fashion over several days following the initial traumatic insult. Apoptotic neurodegeneration contributes in an age-dependent fashion to neuronal injury following head trauma, with the immature brain being exceedingly sensitive. In the most vulnerable ages the apoptosis contribution to the extent of traumatic brain damage far outweighs that of the excitotoxic component.