Brandon A. Miller

Cell Death after Spinal Cord Injury Is Exacerbated by Rapid TNFα-Induced Trafficking of GluR2-Lacking AMPARs to the Plasma Membrane

Glutamate, the major excitatory neurotransmitter in the CNS, is implicated in both normal neurotransmission and excitotoxicity. Numerous in vitro findings indicate that the ionotropic glutamate receptor, AMPAR, can rapidly traffic from intracellular stores to the plasma membrane, altering neuronal excitability. These receptor trafficking events are thought to be involved in CNS plasticity as well as learning and memory. AMPAR trafficking has recently been shown to be regulated by glial release of the proinflammatory cytokine tumor necrosis factor α (TNFα) in vitro. This has potential relevance to several CNS disorders, because many pathological states have a neuroinflammatory component involving TNFα. However, TNFα-induced trafficking of AMPARs has only been explored in primary or slice cultures and has not been demonstrated in preclinical models of CNS damage. Here, we use confocal and image analysis techniques to demonstrate that spinal cord injury (SCI) induces trafficking of AMPARs to the neuronal membrane. We then show that this effect is mimicked by nanoinjections of TNFα, which produces specific trafficking of GluR2-lacking receptors which enhance excitotoxicity. To determine if TNFα-induced trafficking affects neuronal cell death, we sequestered TNFα after SCI using a soluble TNFα receptor, and significantly reduced both AMPAR trafficking and neuronal excitotoxicity in the injury penumbra. The data provide the first evidence linking rapid TNFα-induced AMPAR trafficking to early excitotoxic secondary injury after CNS trauma in vivo, and demonstrate a novel way in which pathological states hijack mechanisms involved in normal synaptic plasticity to produce cell death.

Cerebral protection by hypoxic preconditioning in a murine model of focal ischemia-reperfusion.

Sublethal periods of hypoxia or ischemia can induce adaptive mechanisms to protect against subsequent lethal ischemic insults in a process known as ischemic preconditioning. In the present study, we developed a murine model of cerebral preconditioning using several common strains of adult mice. Animals were exposed to sublethal hypoxia (11% oxygen for 2 h) 48 h prior to a 90 min period of transient focal middle cerebral artery occlusion, induced by an intraluminal filament; injury was assessed 24 h later by TTC staining. Infarct volume in hypoxia-preconditioned animals was reduced 46%, 58%, and 64% in C57Bl/6, 129SvEv, and Swiss-Webster ND4 mice relative to their respective untreated controls. This non-invasive murine model of ischemic tolerance should be useful for elucidating the molecular basis of this protection using transgenic and knockout mice.

Multiparameter Analysis, including EMT Markers, on Negatively Enriched Blood Samples from Patients with Squamous Cell Carcinoma of the Head and Neck

Epithelial to mesenchymal transition (EMT) has been hypothesized as a mechanism by which cells change phenotype during carcinogenesis, as well as tumor metastasis. Whether EMT is involved in cancer metastasis has a specific, practical impact on the field of circulating tumor cells (CTCs). Since the generally accepted definition of a CTC includes the expression of epithelial surface markers, such as EpCAM, if a cancer cell loses its epithelial surface markers (which is suggested in EMT), it will not be separated and/or identified as a CTC. We have developed, and previously reported on the use of, a purely negative enrichment technology enriching for CTCs in the blood of squamous cell carcinoma of the head and neck (SCCHN). This methodology does not depend on the expression of surface epithelial markers. Using this technology, our initial data on SCCHN patient blood indicates that the presence of CTCs correlates with worse disease-free survival. Since our enrichment is not dependent on epithelial markers, we have initiated investigation of the presence of mesenchymal markers in these CTC cells to include analysis of: vimentin, epidermal growth factor receptor, N-cadherin, and CD44. With the aid of confocal microscopy, we have demonstrated not only presumed CTCs that express and/or contain: a nucleus, cytokeratins, vimentin, and either EGFR, CD44, or N-cadherin, but also cells that contain all of the aforementioned proteins except cytokeratins, suggesting that the cells have undergone the EMT process. We suggest that our negative depletion enrichment methodology provides a more objective approach in identifying and evaluating CTCs, as opposed to positive selection approaches, as it is not subjective to a selection bias and can be tailored to accommodate a variety of cytoplasmic and surface markers which can be evaluated to identify a multitude of phenotypic patterns within CTCs from individual patients, including so-called EMT as presented here.

Heterogeneous atypical cell populations are present in blood of metastatic breast cancer patients

IntroductionCirculating tumor cells (CTCs) are commonly isolated from the blood by targeting the epithelial cell adhesion molecule (EpCAM) through positive selection. However, EpCAM can be downregulated during metastatic progression, or it can be initially not present. We designed the present prospective trial to characterize CTCs as well as other circulating cell populations in blood samples from women with metastatic breast cancer without EpCAM-dependent enrichment and/or isolation technology.MethodsA total of 32 patients with metastatic breast cancer were enrolled, and blood samples were processed using a previously described negative depletion immunomagnetic methodology. Samples from healthy volunteers were run as controls (n = 5). Multistep sequential labeling was performed to label and fix cell-surface markers followed by permeabilization for cytokeratins (CK) 8, 18 and 19. Multiparametric flow cytometry (FCM) analysis was conducted using a BD LSR II flow cytometer or a BD FACSAria II or FACSAria III cell sorter. Immunocytochemical staining on postenrichment specimens for DAPI, EpCAM, CD45, CK, epidermal growth factor receptor and vimentin was performed. Expression of these markers was visualized using confocal microscopy (CM).ResultsCD45-negative/CK-positive (CD45− CK+) populations with EpCAM + and EpCAM − expression were identified with both FCM and CM from the negatively enriched patient samples. In addition, EpCAM + and EpCAM − populations that were CK + and coexpressing the pan-hematopoietic marker CD45 were also noted. There were more CK + EpCAM − events/ml than CK + EpCAM + events/ml in both the CD45− and CD45+ fractions (both statistically significant at P ≤ 0.0005). The number of CK + CD45− and CK + CD45+ events per milliliter in blood samples (regardless of EpCAM status) was higher in patient samples than in normal control samples (P ≤ 0.0005 and P ≤ 0.026, respectively). Further, a significant fraction of the CK + CD45+ events also expressed CD68, a marker associated with tumor-associated macrophages. Higher levels of CD45-CK + EpCAM − were associated with worse overall survival (P = 0.0292).ConclusionsMetastatic breast cancer patients have atypical cells that are CK + EpCAM − circulating in their blood. Because a substantial number of these patients do not have EpCAM + CTCs, additional studies are needed to evaluate the role of EpCAM − circulating cells as a prognostic and predictive marker.

Developmental stage of oligodendrocytes determines their response to activated microglia in vitro

BackgroundOligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes are both lost in central nervous system injury and disease. Activated microglia may play a role in OPC and oligodendrocyte loss or replacement, but it is not clear how the responses of OPCs and oligodendrocytes to activated microglia differ.MethodsOPCs and microglia were isolated from rat cortex. OPCs were induced to differentiate into oligodendrocytes with thyroid hormone in defined medium. For selected experiments, microglia were added to OPC or oligodendrocyte cultures. Lipopolysaccharide was used to activate microglia and microglial activation was confirmed by TNFα ELISA. Cell survival was assessed with immunocytochemistry and cell counts. OPC proliferation and oligodendrocyte apoptosis were also assessed.ResultsOPCs and oligodendrocytes displayed phenotypes representative of immature and mature oligodendrocytes, respectively. Activated microglia reduced OPC survival, but increased survival and reduced apoptosis of mature oligodendrocytes. Activated microglia also underwent cell death themselves.ConclusionActivated microglia may have divergent effects on OPCs and mature oligodendrocytes, reducing OPC survival and increasing mature oligodendrocyte survival. This may be of importance because activated microglia are present in several disease states where both OPCs and mature oligodendrocytes are also reacting to injury. Activated microglia may simultaneously have deleterious and helpful effects on different cells after central nervous system injury.

The predictive value of low–field strength magnetic resonance imaging for intraoperative residual tumor detection: Clinical article

OBJECT Neurosurgeons have been utilizing intraoperative MR (iMR) imaging to evaluate the extent of tumor resection since the 1990s. A low-field strength (0.12 T) MR imaging unit (PoleStar N20, Medtronic) is a practical and relatively inexpensive iMR imaging system that has found increased use in neurosurgery. The gold standard for post operative detection of residual tumor has been high-strength MR imaging performed within 48 hours of resection.The object of this study was to determine the predictive concordance of low-strength iMR imaging with standard high-strength MR imaging for detection of residual tumor. METHODS The authors retrospectively evaluated the MR images from 74 intracranial tumor resections, comparing the intraoperative images obtained using a 0.12-T iMR imaging unit to the immediate postoperative images obtained using a standard 1.5-T MR imaging unit within 48 hours after surgery. RESULTS The sensitivity of low-field MR imaging for detection of residual tumor was 0.74 (95% CI 0.58-0.86),and its specificity was 0.97 (95% CI 0.83-1). When only glial tumors (42 of the 74 lesions) were analyzed, the sensitivity was 0.82 (95% CI 0.59-0.94) and the specificity was 0.95 (95% CI 0.73-1). CONCLUSIONS These data could assist the neurosurgeon who has to decide intraoperatively whether the observed iMR images show residual tumor or not.

Tumor necrosis factor alpha mediates GABA(A) receptor trafficking to the plasma membrane of spinal cord neurons in vivo.

The proinflammatory cytokine TNFα contributes to cell death in central nervous system (CNS) disorders by altering synaptic neurotransmission. TNFα contributes to excitotoxicity by increasing GluA2-lacking AMPA receptor (AMPAR) trafficking to the neuronal plasma membrane. In vitro, increased AMPAR on the neuronal surface after TNFα exposure is associated with a rapid internalization of GABAA receptors (GABAARs), suggesting complex timing and dose dependency of the CNSs response to TNFα. However, the effect of TNFα on GABAAR trafficking in vivo remains unclear. We assessed the effect of TNFα nanoinjection on rapid GABAAR changes in rats (N = 30) using subcellular fractionation, quantitative western blotting, and confocal microscopy. GABAAR protein levels in membrane fractions of TNFα and vehicle-treated subjects were not significantly different by Western Blot, yet high-resolution quantitative confocal imaging revealed that TNFα induces GABAAR trafficking to synapses in a dose-dependent manner by 60 min. TNFα-mediated GABAAR trafficking represents a novel target for CNS excitotoxicity.

Surgical technique for spinal cord delivery of therapies: demonstration of procedure in gottingen minipigs.

This is a compact visual description of a combination of surgical technique and device for the delivery of (gene and cell) therapies into the spinal cord. While the technique is demonstrated in the animal, the procedure is FDA-approved and currently being used for stem cell transplantation into the spinal cords of patients with ALS. While the FDA has recognized proof-of-principle data on therapeutic efficacy in highly characterized rodent models, the use of large animals is considered critical for validating the combination of a surgical procedure, a device, and the safety of a final therapy for human use. The size, anatomy, and general vulnerability of the spine and spinal cord of the swine are recognized to better model the human. Moreover, the surgical process of exposing and manipulating the spinal cord as well as closing the wound in the pig is virtually indistinguishable from the human. We believe that the healthy pig model represents a critical first step in the study of procedural safety.

A Sublethal Dose of TNFα Potentiates Kainate-Induced Excitotoxicity in Optic Nerve Oligodendrocytes

Glutamate receptor-induced cell death, known as excitotoxicity in both neurons and oligodendrocytes, has been implicated as a common pathway of cell death in numerous central nervous system (CNS) diseases and trauma. Research in both neuronal and oligodendrocyte excitotoxicity has examined glutamate’s receptor-mediated effects on CNS cells, and explored strategies to protect cells exposed to the elevated glutamate levels that occur in CNS trauma and disease. Proinflammatory cytokines are also elevated in the injured CNS, and have also been implicated in CNS cell death. Recently, several laboratories have examined cytokines’ effects on neuronal and glial excitotoxicity. Here, we review literature concerning the dynamic susceptibility of both neurons and oligodendrocytes to excitotoxicity, and present new data from our laboratory showing that the susceptibility of oligodendrocytes to excitotoxicity is acutely potentiated by the proinflammatory cytokine TNFα.

Mild traumatic brain injury in children is associated with a low risk for posttraumatic seizures

OBJECT Of the 1.7 million traumatic brain injuries (TBIs) in the US, a third occur in patients under 14 years of age. The rate of posttraumatic epilepsy (PTE) may be as high as 19% after severe pediatric TBI, but the risk for seizures after mild TBI is unknown. Although the rate of seizures after mild TBI may be low, current practice is often driven by high clinical concern for posttraumatic seizures. In this study, the authors evaluated electroencephalography (EEG) results and antiepileptic drug (AED) use in a large cohort of children with mild TBI to estimate the incidence of posttraumatic seizures in this population. METHODS Patients presenting to Childrens Hospital of Atlanta for mild TBI from 2010 to 2013 were evaluated. Five thousand one hundred forty-eight patients with mild TBI were studied and divided into 3 groups: 4168 who were discharged from the emergency department, 868 who were admitted without neurosurgical intervention, and 112 who underwent neurosurgical procedures (craniotomy for hematoma evacuation or elevation of depressed skull fractures) but were discharged without an extended stay. Demographic information, CT characteristics, EEG reports, and prescriptions for AEDs were analyzed. Long-term follow-up was sought for all patients who underwent EEG. Correlation between EEG result and AED use was also evaluated. RESULTS All patients underwent head CT, and admitted patients were more likely to have an abnormal study (p < 0.0001). EEG evaluations were performed for less than 1.0% of patients in all 3 categories, without significant differences between groups (p = 0.97). Clinicians prescribed AEDs in less than 2.0% of patients for all groups, without significant differences between groups (p = 0.094). Even fewer children continue to see a neurologist for long-term seizure management. The EEG result had good negative predictive value, but only an abnormal EEG reading that was diagnostic of seizures correlated significantly with AED prescription (p = 0.04). CONCLUSIONS EEG utilization and AED prescription was low in all 3 groups, indicating that seizures following mild TBI are likely rare events. EEG has good negative predictive value for patients who did not receive AEDs, but has poorer positive predictive value for AED use.