David D. Limbrick

Resting-state activity in development and maintenance of normal brain function

One of the most intriguing recent discoveries concerning brain function is that intrinsic neuronal activity manifests as spontaneous fluctuations of the blood oxygen level–dependent (BOLD) functional MRI signal. These BOLD fluctuations exhibit temporal synchrony within widely distributed brain regions known as resting-state networks. Resting-state networks are present in the waking state, during sleep, and under general anesthesia, suggesting that spontaneous neuronal activity plays a fundamental role in brain function. Despite its ubiquitous presence, the physiological role of correlated, spontaneous neuronal activity remains poorly understood. One hypothesis is that this activity is critical for the development of synaptic connections and maintenance of synaptic homeostasis. We had a unique opportunity to test this hypothesis in a 5-y-old boy with severe epileptic encephalopathy. The child developed marked neurologic dysfunction in association with a seizure disorder, resulting in a 1-y period of behavioral regression and progressive loss of developmental milestones. His EEG showed a markedly abnormal pattern of high-amplitude, disorganized slow activity with frequent generalized and multifocal epileptiform discharges. Resting-state functional connectivity MRI showed reduced BOLD fluctuations and a pervasive lack of normal connectivity. The child underwent successful corpus callosotomy surgery for treatment of drop seizures. Postoperatively, the patients behavior returned to baseline, and he resumed development of new skills. The waking EEG revealed a normal background, and functional connectivity MRI demonstrated restoration of functional connectivity architecture. These results provide evidence that intrinsic, coherent neuronal signaling may be essential to the development and maintenance of the brains functional organization.

In vitro status epilepticus causes sustained elevation of intracellular calcium levels in hippocampal neurons.

Calcium ions and calcium-dependent systems have been implicated in the pathophysiology of status epilepticus (SE). However, the dynamics of intracellular calcium ([Ca2+]i) levels during SE has not yet been studied. We have employed the hippocampal neuronal culture (HNC) model of in vitro SE that produces continuous epileptiform discharges to study spatial and dynamic changes in [Ca2+]i levels utilizing confocal laser scanning microscopy and the calcium binding dye, indo-1. During SE, the average [Ca2+]i levels increased from control levels of 150-200 nM to levels of 450-600 nM. This increased [Ca2+]i was maintained for the duration of SE. Following SE, [Ca2+]i levels gradually returned to basal values. The duration of SE was shown to affect the ability of the neuron to restore resting [Ca2+]i levels. Both N-methyl-D-aspartate (NMDA) receptor-gated and voltage-gated Ca2+ channels (VGCCs) contributed to the increased calcium entry during SE. Moreover, this elevation in [Ca2+]i occurred in both the nucleus and cytosol. These results provide the first dynamic measurement of [Ca2+]i during prolonged electrographic seizure discharges in an in vitro SE model and suggest that prolonged epileptiform discharges give rise to abnormal sustained increases in [Ca2+]i levels that may play a role in the neuronal cell damage and long-term plasticity changes associated with SE.

Inability to restore resting intracellular calcium levels as an early indicator of delayed neuronal cell death

The hippocampus is especially vulnerable to excitotoxicity and delayed neuronal cell death. Chronic elevations in free intracellular calcium concentration ([Ca2+]i) following glutamate-induced excitotoxicity have been implicated in contributing to delayed neuronal cell death. However, no direct correlation between delayed cell death and prolonged increases in [Ca2+]i has been determined in mature hippocampal neurons in culture. This investigation was initiated to determine the statistical relationship between delayed neuronal cell death and prolonged increases in [Ca2+]i in mature hippocampal neurons in culture. Using indo-1 confocal fluorescence microscopy, we observed that glutamate induced a rapid increase in [Ca2+]i that persisted after the removal of glutamate. Following excitotoxic glutamate exposure, neurons exhibited prolonged increases in [Ca2+]i, and significant delayed neuronal cell death was observed. The N-methyl-D-aspartate (NMDA) channel antagonist MK-801 blocked the prolonged increases in [Ca2+]i and cell death. Depolarization of neurons with potassium chloride (KCl) resulted in increases in [Ca2+]i, but these increases were buffered immediately upon removal of the KCl, and no cell death occurred. Linear regression analysis revealed a strong correlation (R = 0.973) between glutamate-induced prolonged increases in [Ca2+]i and delayed cell death. These data suggest that excitotoxic glutamate exposure results in an NMDA-induced inability to restore resting [Ca2+]i (IRRC) that is a statistically significant indicator of delayed neuronal cell death.

Targeted Inhibition of Cyclic AMP Phosphodiesterase-4 Promotes Brain Tumor Regression

Purpose: As favorable outcomes from malignant brain tumors remain limited by poor survival and treatment-related toxicity, novel approaches to cure are essential. Previously, we identified the cyclic AMP phosphodiesterase-4 (PDE4) inhibitor Rolipram as a potent antitumor agent. Here, we investigate the role of PDE4 in brain tumors and examine the utility of PDE4 as a therapeutic target. Experimental Design: Immunohistochemistry was used to evaluate the expression pattern of a subfamily of PDE4, PDE4A, in multiple brain tumor types. To evaluate the effect of PDE4A on growth, a brain-specific isoform, PDE4A1 was overexpressed in xenografts of Daoy medulloblastoma and U87 glioblastoma cells. To determine therapeutic potential of PDE4 inhibition, Rolipram, temozolomide, and radiation were tested alone and in combination on mice bearing intracranial U87 xenografts. Results: We found that PDE4A is expressed in medulloblastoma, glioblastoma, oligodendroglioma, ependymoma, and meningioma. Moreover, when PDE4A1 was overexpressed in Daoy medulloblastoma and U87 glioblastoma cells, in vivo doubling times were significantly shorter for PDE4A1-overexpressing xenografts compared with controls. In long-term survival and bioluminescence studies, Rolipram in combination with first-line therapy for malignant gliomas (temozolomide and conformal radiation therapy) enhanced the survival of mice bearing intracranial xenografts of U87 glioblastoma cells. Bioluminescence imaging indicated that whereas temozolomide and radiation therapy arrested intracranial tumor growth, the addition of Rolipram to this regimen resulted in tumor regression. Conclusions: This study shows that PDE4 is widely expressed in brain tumors and promotes their growth and that inhibition with Rolipram overcomes tumor resistance and mediates tumor regression.

Hydrocephalus in children

Hydrocephalus is a common disorder of cerebral spinal fluid (CSF) physiology resulting in abnormal expansion of the cerebral ventricles. Infants commonly present with progressive macrocephaly whereas children older than 2 years generally present with signs and symptoms of intracranial hypertension. The classic understanding of hydrocephalus as the result of obstruction to bulk flow of CSF is evolving to models that incorporate dysfunctional cerebral pulsations, brain compliance, and newly characterised water-transport mechanisms. Hydrocephalus has many causes. Congenital hydrocephalus, most commonly involving aqueduct stenosis, has been linked to genes that regulate brain growth and development. Hydrocephalus can also be acquired, mostly from pathological processes that affect ventricular outflow, subarachnoid space function, or cerebral venous compliance. Treatment options include shunt and endoscopic approaches, which should be individualised to the child. The long-term outcome for children that have received treatment for hydrocephalus varies. Advances in brain imaging, technology, and understanding of the pathophysiology should ultimately lead to improved treatment of the disorder.

Endoscopic third ventriculostomy and choroid plexus cauterization in infants with hydrocephalus: a retrospective Hydrocephalus Clinical Research Network study

OBJECT The use of endoscopic third ventriculostomy (ETV) with choroid plexus cauterization (CPC) has been advocated as an alternative to CSF shunting in infants with hydrocephalus. There are limited reports of this procedure in the North American population, however. The authors provide a retrospective review of the experience with combined ETV + CPC within the North American Hydrocephalus Clinical Research Network (HCRN). METHODS All children (< 2 years old) who underwent an ETV + CPC at one of 7 HCRN centers before November 2012 were included. Data were collected retrospectively through review of hospital records and the HCRN registry. Comparisons were made to a contemporaneous cohort of 758 children who received their first shunt at < 2 years of age within the HCRN. RESULTS Thirty-six patients with ETV + CPC were included (13 with previous shunt). The etiologies of hydrocephalus were as follows: intraventricular hemorrhage of prematurity (9 patients), aqueductal stenosis (8), myelomeningocele (4), and other (15). There were no major intraoperative or early postoperative complications. There were 2 postoperative CSF infections. There were 2 deaths unrelated to hydrocephalus and 1 death from seizure. In 18 patients ETV + CPC failed at a median time of 30 days after surgery (range 4-484 days). The actuarial 3-, 6-, and 12-month success for ETV + CPC was 58%, 52%, and 52%. Time to treatment failure was slightly worse for the 36 patients with ETV + CPC compared with the 758 infants treated with shunts (p = 0.012). Near-complete CPC (≥ 90%) was achieved in 11 cases (31%) overall, but in 50% (10 of 20 cases) in 2012 versus 6% (1 of 16 cases) before 2012 (p = 0.009). Failure was higher in children with < 90% CPC (HR 4.39, 95% CI 0.999-19.2, p = 0.0501). CONCLUSIONS The early North American multicenter experience with ETV + CPC in infants demonstrates that the procedure has reasonable safety in selected cases. The degree of CPC achieved might be associated with a surgeons learning curve and appears to affect success, suggesting that surgeon training might improve results.

Calcium influx constitutes the ionic basis for the maintenance of glutamate-induced extended neuronal depolarization associated with hippocampal neuronal death.

Excessive activation of neuronal glutamate receptors has been implicated in the pathophysiology of stroke, epilepsy, and traumatic brain injury. Previously, it has been demonstrated that excitotoxic glutamate exposure results in the induction of an extended neuronal depolarization (END), as well as protracted elevations in free intracellular calcium ([Ca(2+)](i)). Both END and the prolonged [Ca(2+)](i) elevations were shown to correlate with subsequent neuronal death. In the current study, we used whole-cell current-clamp electrophysiology and fura-ff Ca(2+) imaging to determine the electrophysiological basis of END. We found that removal of extracellular Ca(2+) but not Na(+) in the post-glutamate period resulted in complete reversal of END, allowing neurons to rapidly repolarize to their initial resting membrane potential (RMP). In addition, removal of extracellular Ca(2+) was sufficient to eliminate the protracted [Ca(2+)](i) elevations induced by excitotoxic glutamate exposure. To investigate the mechanism through which extracellular Ca(2+) was effecting these changes, pharmacological antagonists of well-characterized routes of Ca(2+) entry were tested for their effects on END. Antagonists of glutamate receptors and voltage-gated Ca(2+) channels (VGCCs) had no significant effect on the membrane potential of neurons in END. Likewise, inhibitors of the Na(+)/Ca(2+) exchange (NCX) were ineffective. In contrast, addition of 500 microM ZnCl(2) or 100 microM GdCl(3) to control extracellular medium (containing normal levels of extracellular Ca(2+)) in the post-glutamate period resulted in rapid and complete reversal of END. Addition of 1mM CdCl(2) to control medium had only modest effects on END. These data provide the first direct evidence that END induced by excitotoxic glutamate exposure is caused by an influx of extracellular Ca(2+) and demonstrate that the previously irreversible condition of END can be reversed by removing extracellular Ca(2+). In addition, understanding the electrophysiological basis of this novel Ca(2+)-induced extended depolarization may provide an insight into the pathophysiology of stroke, traumatic brain injury, and other forms of neuronal injury.

Hemispherotomy: efficacy and analysis of seizure recurrence

OBJECT Hemispherotomy generally is performed in hemiparetic patients with severe, intractable epilepsy arising from one cerebral hemisphere. In this study, the authors evaluate the efficacy of hemispherotomy and present an analysis of the factors influencing seizure recurrence following the operation. METHODS The authors performed a retrospective review of 49 patients (ages 0.2-20.5 years) who underwent functional hemispherotomy at their institution. The first 14 cases were traditional functional hemispherotomies, and included temporal lobectomy, while the latter 35 were performed using a modified periinsular technique that the authors adopted in 2003. RESULTS Thirty-eight of the 49 patients (77.6%) were seizure free at the termination of the study (mean follow-up 28.6 months). Of the 11 patients who were not seizure free, all had significant improvement in seizure frequency, with 6 patients (12.2%) achieving Engel Class II outcome and 5 patients (10.2%) achieving Engel Class III. There were no cases of Engel Class IV outcome. The effect of hemispherotomy was durable over time with no significant change in Engel class over the postoperative follow-up period. There was no statistical difference in outcome between surgery types. Analysis of factors contributing to seizure recurrence after hemispherotomy revealed no statistically significant predictors of treatment failure, although bilateral electrographic abnormalities on the preoperative electroencephalogram demonstrated a trend toward a worse outcome. CONCLUSIONS In the present study, hemispherotomy resulted in freedom from seizures in nearly 78% of patients; worthwhile improvement was demonstrated in all patients. The seizure reduction observed after hemispherotomy was durable over time, with only rare late failure. Bilateral electrographic abnormalities may be predictive of posthemispherotomy recurrent seizures.

Effect of implementation of a paediatric neurocritical care programme on outcomes after severe traumatic brain injury: a retrospective cohort study

BACKGROUND Outcomes after traumatic brain injury are worsened by secondary insults; modern intensive-care units address such challenges through use of best-practice pathways. Organisation of intensive-care units has an important role in pathway effectiveness. We aimed to assess the effect of a paediatric neurocritical care programme (PNCP) on outcomes for children with severe traumatic brain injury. METHODS We undertook a retrospective cohort study of 123 paediatric patients with severe traumatic brain injury (Glasgow coma scale scores ≤8, without gunshot or abusive head trauma, cardiac arrest, or Glasgow coma scale scores of 3 with fixed and dilated pupils) admitted to the paediatric intensive-care unit of the St Louis Childrens Hospital (St Louis, MO, USA) between July 15, 1999, and Jan 15, 2012. The primary outcome was rate of categorised hospital discharge disposition before and after implementation of a PNCP on Sept 17, 2005. We developed an ordered probit statistical model to assess adjusted outcome as a function of initial injury severity. We assessed care-team behaviour by comparing timing of invasive neuromonitoring and scored intensity of therapies targeting intracranial hypertension. FINDINGS Characteristics of treated patients (aged 3-219 months) were much the same between treatment periods. Before PNCP implementation, 33 (52%) of 63 patients had unfavourable disposition at hospital discharge (death or admission to an inpatient facility) and 30 (48%) had a favourable disposition (home with or without treatment); after PNCP implementation, 20 (33%) of 60 patients had unfavourable disposition and 40 (67%) had favourable disposition (p=0·01). Seven (11%) patients died before PNCP implementation compared with two (3%) deaths after implementation. The probit model indicated that outcome improved across the spectrum of Glasgow coma scale scores after resuscitation (p=0·02); this improvement progressed with increasing injury severity. Kaplan-Meier analysis suggested that neuromonitoring was started earlier and maintained longer after implementation of the PNCP (p=0·03). Therapeutic intensity scores were increased for the first 3 days of treatment after PNCP implementation (p=0·0298 for day 1, p=0·0292 for day 2, and p=0·0471 for day 3). The probit model suggested that increasing age (p=0·03), paediatric risk of mortality III scores (p=0·0003), and injury severity scores (p=0·02) were reliably associated with increased probability of unfavourable outcomes whereas white race (p=0·01), use of intracranial pressure monitoring (p=0·001), and increasing Glasgow coma scale scores after resuscitation (p=0·04) were associated with increased probability of favourable outcomes. INTERPRETATION Outcomes for children with traumatic brain injury can be improved by altering the care system in a way that stably implements a cooperative programme of accepted best practice. FUNDING St Louis Childrens Hospital and the Sean Glanvill Foundations.

Impact of epilepsy surgery on seizure control and quality of life: A 26-year follow-up study

Purpose:  The short‐term efficacy and safety of epilepsy surgery relative to medical therapy has been established, but it remains underutilized. There is a lack of data regarding the long‐term seizure‐control rates and quality of life outcomes after epilepsy surgery. This study represents the longest follow‐up study to date, with a mean follow‐up duration of 26 years.