Faculty Bibliography

OBJECTIVE: Intraoperative identification of cerebrospinal fluid (CSF) leakage is critical in successful closure after endoscopic cranial base surgery. Intrathecal injection of fluorescein is quite useful in identifying CSF leaks. However, complications have been reported with various doses and the technique has fallen out of favor. We explored the safety of low-dose intrathecal fluorescein administered to patients undergoing endoscopic cranial base surgery. METHODS: A retrospective chart review and postoperative patient survey were performed. The nature and incidence of complications and subjective complaints were recorded in 54 patients who underwent endoscopic, endonasal approaches to the anterior cranial base and received intrathecal fluorescein after premedication with dexamethasone and diphenhydramine. RESULTS: Intraoperative CSF leak was identified with fluorescein in 46.3% of the patients and helped determine the reconstruction technique. Postoperative CSF leak occurred in 9.3% of the patients and resolved with lumbar drainage. There were no seizures. Most side effects were nonspecific, transient, and likely not caused by fluorescein including malaise (57.4%), headache (51.9%), dizziness (31.5%), or nausea/vomiting (24.1%). Three patients (5.6%) experienced persistent subjective lower extremity weakness (n = 2) and numbness (n = 2) postoperatively; however, two of them had undergone lumbar drainage. CONCLUSION: Low-dose injection of intrathecal fluorescein after premedication with steroid and antihistamine agents is generally safe. Most symptoms are nonspecific and transient, likely caused by the surgery or lumbar drainage. However, fluorescein should be administered with some caution because it may be responsible for occasional lower extremity weakness and numbness

OBJECTIVES: Reconstruction following endoscopic skull base surgery requires a high degree of success to avoid the morbidity of postoperative cerebrospinal fluid (CSF) leak. The impact on outcomes of CSF visualization with intrathecal fluorescein, however, is unknown. STUDY DESIGN: A retrospective review of patients undergoing endoscopic skull base surgery with intrathecal fluorescein. A possible correlation between intraoperative fluorescein identification and postoperative CSF leak was analyzed. RESULTS: 61 patients underwent surgery for a variety of lesions including pituitary adenoma (55.7%), encephalocele (14.8%), and meningioma (9.8%). Seven (19.4%) of the 37 patients with intraoperative fluorescein leak experienced postoperative CSF leak compared to 0 of the 24 patients who did not have intraoperative fluorescein leak (P = 0.02). All cases of CSF leak resolved with lumbar drainage alone. CONCLUSIONS: The lack of intraoperative fluorescein leakage correlates strongly with a low risk for postoperative CSF leak. This can be used to stratify the extent of skull base reconstruction required during endoscopic skull base surgeries

Intradiploic cerebrospinal fluid (CSF) collections represent a rare complication of either head trauma or neurosurgical procedures. Their formation is thought to depend partly on violation of the meninges and the inner cortical bone abutting the neuraxis. The authors present two pediatric cases involving diploic CSF collections following neurosurgical interventions. In the first case, a CSF fistula was found within the occipital bone and the petrous portion of the temporal bone 9 years after a suboccipital craniectomy. The second case features the extremely rare occurrence of a CSF fistula within a thoracic vertebra, 9 years after a laminectomy. Both patients underwent successful surgical procedures for repair of the fistulas

Do continuous subthreshold oscillations in membrane potential within an electrically coupled network depend on gap junctional coupling? For the inferior olive (IO), modeling and developmental studies suggested that the answer is yes, although physiological studies of connexin36 knock-out mice lacking electrical coupling suggested that the answer is no. Here we addressed the question differently by using a lentivirus-based vector to express, in the IO of adult rats, a single amino acid mutation of connexin36 that disrupts the intracellular trafficking of wild-type connexin36 and blocks gap junctional coupling. Confocal microscopy of green fluorescence protein-labeled dendrites revealed that the mutant connexin36 prevented wild-type connexin36 from being expressed in dendritic spines of IO neurons. Intracellular recordings from lentivirally transduced IO networks revealed that robust and continuous subthreshold oscillations require gap junctional coupling of IO neuron somata within 40 microm of one another. Topological studies indicated that the minimal coupled network for supporting such oscillations may be confined to the dendritic arbor of a single IO neuron. Occasionally, genetically uncoupled IO neurons showed transient oscillations; however, these were not sustained longer than 3 s and were 69% slower and 71% smaller than the oscillations of normal IO neurons, a finding replicated with carbenoxolone, a pharmacological antagonist of gap junctions. The experiments provided the first direct evidence that gap junctional coupling between neurons, specifically mediated by connexin36, allows a continuous network oscillation to emerge from a population of weak and episodic single-cell oscillators. The findings are discussed in the context of the importance of gap junctions for cerebellar rhythms involved in movement

The hypothesis is presented that a disruption in brain synchronization contributes to autism by destroying the coherence of brain rhythms and slowing overall cognitive processing speed. Particular focus is on the inferior olive, a precerebellar structure that is reliably disrupted in autism and which normally generates a coherent 5-13 Hz rhythmic output. New electrophysiological data reveal that the continuity of the rhythmical oscillation in membrane potential generated by inferior olive neurons requires the formation of neuronal assemblies by the connexin36 protein that mediates electrical synapses and promotes neuronal synchrony. An experiment with classical eyeblink conditioning is presented to demonstrate that the inferior olive is necessary to learn about sequences of stimuli presented at intervals in the range of 250-500 ms, but not at 700 ms, revealing that a disruption of the inferior olive slows stimulus processing speed on the time scale that is lost in autistic children. A model is presented in which the voltage oscillation generated by populations of electrically synchronized inferior olivary neurons permits the utilization of sequences of stimuli given at, or faster than, 2 per second. It is expected that the disturbance in inferior olive structure in autism disrupts the ability of inferior olive neurons to become electrically synchronized and to generate coherent rhythmic output, thereby impairing the ability to use rapid sequences of cues for the development of normal language skill. Future directions to test the hypothesis are presented

Inferior olive (IO) neurons are electrically coupled by cytosolic pores formed by the neuron-specific connexin 36 (Cx36). Electrical coupling in the IO figures prominently in current views about brain control of movement. However, a role for Cx36 in movement has been questioned and not definitively demonstrated. Previous reports have shown that embryonic deletion of the Cx36 gene resulted in almost complete loss of cytosolic and electrical coupling in the IO without an obvious deficit in movement, possibly due to developmental compensations in ionic conductances that can confound the approach of embryonic gene deletion. We used a replication-incompetent lentiviral vector to stably express a dominant-negative Cx36 mutant in the IO of adult rats. We show that interneuronal cytosolic coupling is severely reduced by the mutant Cx36, without effect on neuron morphology or electrical properties. Multisite electromyography revealed that blocking Cx36 in the IO impaired the coherence of muscle firing during harmaline tremor without affecting its rhythm. The data demonstrate that gap junction coupling within the IO mediated by Cx36 adds 10-20 ms of precision to the fine temporal coordination of muscle firing during movement

Rat connexin 36 (Cx36) was mutated by substituting serine for cysteine at residue 231 (C231S) and the mutant's effect on the subcellular localization of wild-type Cx36 and the intercellular permeability that it confers was determined in human HeLa and rat PC12 cells. Cells transfected with the mutant or wild-type Cx36 cDNA expressed the expected 36 kDa protein and Cx36 immunoreactivity. Co-immunoprecipitation experiments with monkey COS-7 cells transiently transfected with both mutant and wild-type Cx36 cDNAs demonstrated that the mutant protein bound to the wild-type. Double immunofluorescence microscopy of stably transfected HeLa cells demonstrated that mutant Cx36 blocked the transport of the wild-type Cx36 to the cell membrane, primarily by trapping it in the endoplasmic reticulum around the nucleus. Coexpression of the mutant Cx36 with the wild-type protein abolished the ability of the latter to permit dye transfer in both HeLa and PC12 cells. The findings are the first demonstration of a mutation of Cx36 that inhibits wild-type Cx36 function in mammalian cells

The experiments strongly suggested that the reason why Purkinje cells die so easily after global brain ischemia relates to deficiencies in aldolase C and EAAT4 that allow them to survive pathologically intense synaptic input from the inferior olive after the restoration of blood flow. This conclusion is based on: (a) the remarkably tight correspondence between the regional absence of aldolase C and EAAT4 in Purkinje cells and the patterned loss of Purkinje cells after a bout of global brain ischemia; (b) the necessity of the olivocerebellar pathway for the ischemic death of Purkinje cells; and (c) the build-up of pathologically synchronous and high-frequency burst activity within the inferior olive during recovery from ischemia. Indeed, the correspondence between the absence of aldolase C and EAAT4 to sensitivity to ischemia could be demonstrated for zones of Purkinje cells as small as two neurons. A second finding was that Purkinje cells are not uniformly sensitive to transient ischemia, since they die most frequently in zones where aldolase C and EAAT4 are absent. One implication of the experiment is that factors beyond the unique synaptic and membrane properties of Purkinje cells play an important role in determining this neuron's high sensitivity to ischemia. The data strongly imply that two properties of Purkinje cells that make them susceptible to ischemic death are their reduced capability to sequester glutamate and reduced ability to generate energy during anoxia. The patterned death of Purkinje cells is sufficient to induce a form of audiogenic myoclonus, as determined with a neurotoxic dose of ibogaine. Ibogaine-induced myoclonus is recognized behaviorally as a reduced ability to habituate to a startle stimulus and resembles the myoclonic jerk of rats during recovery from a prolonged bout of global brain ischemia. Commonalities of ischemia and ibogaine-induced neurodegeneration are the intricately striped Purkinje cell loss in the posterior lobe and a nearly complete deafferentation of the lateral aspect of the fastigial nucleus from the cerebellar cortex, in particular the dorsolateral protuberance. Thus, the data point strongly to a cerebellar contribution to audiogenic myoclonus. Single-neuron electrophysiology experiments in monkeys have demonstrated that the evoked activity in the deep cerebellar nuclei occurs too late to initiate the startle response (60) and electromyography of the postischemic myoclonus of rats corroborates this view (see Chapter 31) (20). However, the nearly complete loss of GABAergic terminals in the dorsolateral protuberance after Purkinje cell death would be expected to dramatically increase its tonic firing and the background excitation of the brain-stem structures that it innervates. The fastigial nucleus innervates a large number of autonomic and motor structures in the brainstem and diencephalon, including the ventrolateral nucleus of the thalamus and the gigantocellular reticular nucleus in the medulla--structures that have been implicated in human posthypoxic myoclonus (6, 7). We propose that the posthypoxic myoclonic jerk of rats is, at least in part, due to disinhibition of the fastigial nucleus produced by patterned Purkinje cell death in the vermis. The argument is as follows: the loss of GABAergic inhibition in the fastigial nucleus after ischemia leads to diaschisis of the motor thalamus and reticular formation which, in turn, is responsible for enhanced motor excitability and myoclonus. That the audiogenic myoclonus after global brain ischemia in the rat gradually resolves over a period of 2 to 3 weeks is consistent with this view, as restoration of background excitability after CNS damage in rats has been documented to occur within this time-frame (61). Our view brings together the physiologic finding that posthypoxic myoclonus appears to originate in the sensory-motor cortices and/or reticular formation with the consistent anatomical finding of Purkinje cell loss after ischemia, and explains the puzzle of Marsden's unique cases of myoclonus associated with coeliac disease (1). Moreover, our argument is consistent with findings both in rats (62, 63) and humans (64) that damage to the vermis impairs the long-term habituation of the startle reflex. It remains to be determined whether the pathologically enhanced startle responses after vermal damage resemble brain-stem reticular or cortical myoclonus at the electrophysiologic level of analysis. What is the purpose of the regional expression of aldolase C and EAAT4 in Purkinje cells? The close correspondence between the spatial distribution of aldolase C and the parasagittal anatomy of the cerebellum (48) has led to the view that aldolase C may help specify connectivity during development. While the present experiments do not address this issue, they underscore the fact that aldolase plays a fundamental role in metabolism. Because Purkinje cells have a repressed expression of aldolase A (31), whatever role the absence of aldolase C may play during development comes at the price of metabolic frailty later in adulthood. From another point of view, aldolase C and EAAT4 appear to confer upon Purkinje cells the ability to survive their own climbing fiber. Indeed, climbing fibers form a distributed synapse that synchronously releases glutamate (or aspartate) at all levels of the dendritic tree simultaneously (65, 66). Such synchronous activation triggers calcium influx throughout the Purkinje cell dendrites at a magnitude that is unparalleled in the nervous system (12), and, thus, places an extraordinarily high metabolic demand on the Purkinje cell. The apparently reduced level of aldolase in a subpopulation of Purkinje cells provides the condition for energy failure and death during anoxia so long as the climbing fibers are intact or when climbing fiber activation is pharmacologically enhanced under normoxic conditions, such as after ibogaine (53-56). Lastly, the argument that diaschisis produced by patterned cerebellar degeneration leads to thalamo-cortical and reticular hyperexcitability agrees with C. David Marsden and his colleagues' bold demonstration of an inhibitory influence of cerebellar cortex on motor cortex in humans (67). Our anatomic data indicate that the spatially distinct zones of Purkinje cells, which are killed by global brain ischemia, may be the origin of such inhibition