Gene expression patterns following unilateral traumatic brain injury reveals a local pro-inflammatory and remote anti-inflammatory response.

BackgroundTraumatic brain injury (TBI) results in irreversible damage at the site of impact and initiates cellular and molecular processes that lead to secondary neural injury in the surrounding tissue. We used microarray analysis to determine which genes, pathways and networks were significantly altered using a rat model of TBI. Adult rats received a unilateral controlled cortical impact (CCI) and were sacrificed 24 h post-injury. The ipsilateral hemi-brain tissue at the site of the injury, the corresponding contralateral hemi-brain tissue, and naïve (control) brain tissue were used for microarray analysis. Ingenuity Pathway Analysis (IPA) software was used to identify molecular pathways and networks that were associated with the altered gene expression in brain tissues following TBI.ResultsInspection of the top fifteen biological functions in IPA associated with TBI in the ipsilateral tissues revealed that all had an inflammatory component. IPA analysis also indicated that inflammatory genes were altered on the contralateral side, but many of the genes were inversely expressed compared to the ipsilateral side. The contralateral gene expression pattern suggests a remote anti-inflammatory molecular response. We created a network of the inversely expressed common (i.e., same gene changed on both sides of the brain) inflammatory response (IR) genes and those IR genes included in pathways and networks identified by IPA that changed on only one side. We ranked the genes by the number of direct connections each had in the network, creating a gene interaction hierarchy (GIH). Two well characterized signaling pathways, toll-like receptor/NF-kappaB signaling and JAK/STAT signaling, were prominent in our GIH.ConclusionsBioinformatic analysis of microarray data following TBI identified key molecular pathways and networks associated with neural injury following TBI. The GIH created here provides a starting point for investigating therapeutic targets in a ranked order that is somewhat different than what has been presented previously. In addition to being a vehicle for identifying potential targets for post-TBI therapeutic strategies, our findings can also provide a context for evaluating the potential of therapeutic agents currently in development

Randomized, Placebo-Controlled, Double-Blind Pilot Study of D-Cycloserine in Chronic Stroke

Stroke is a leading cause of death and disability in the USA. Up to 60% of patients do not fully recover despite intensive physical therapy treatment. N-Methyl-D-aspartate receptors (NMDA-R) have been shown to play a role in synaptic plasticity when activated. D-Cycloserine promotes NMDA receptor function by binding to receptors with unoccupied glycine sites. These receptors are involved in learning and memory. We hypothesized that D-cycloserine, when combined with robotic-assisted physiotherapy (RAP), would result in greater gains compared with placebo + RAP in stroke survivors. Participants (n=14) were randomized to D-cycloserine plus RAP or placebo plus RAP. Functional, cognitive, and quality-of-life measures were used to assess recovery. There was significant improvement in grip strength of the affected hand within both groups from baseline to 3 weeks (95% confidence interval for mean change, 3.95 ± 2.96 to 4.90 ± 3.56 N for D-cycloserine and 5.72 ± 3.98 to 8.44 ± 4.90 N for control). SIS mood domain showed improvement for both groups (95% confidence interval for mean change, 72.6 ± 16.3 to 82.9 ± 10.9 for D-cycloserine and 82.9 ± 13.5 to 90.3 ± 9.9 for control). This preliminary study does not provide evidence that D-cycloserine can provide greater gains in learning compared with placebo for stroke survivors

Deformation response and cytosolic calcium increases in NTera2 neurons subjected to traumatic levels of hydrodynamic shear stress

A biomechanical method was developed to model traumatic brain injury in vitro by hydrodynamically deforming NTera2 neurons while monitoring intracellular free calcium concentration ( (Ca\sp{2+}\rbrack\sb{\rm i}). Fluid shear stress caused membrane strain rates greater than 1 s\sp{-1}, loading conditions which mimic inertial traumatic loading. Cellular strain, measured by tracking membrane-bound microspheres, ranged from 0 to 52%. \rm\lbrack Ca\sp{2+}\rbrack\sb{i}, assesses by Fura-2, increased with strain and loading rate. Furthermore, \rm\lbrack Ca\sp{2+}\rbrack\sb{i} increases correlated with strain (R\sp2 = 0.75), suggesting a deformation induced injury. Leakage of cytosolic lactate dehydrogenase (LDH) was used to assess cell viability in the first 24 hr following injury. Acute ($\u3c$5 min) LDH release correlated with loading rate (R\sp2 = 0.81) and, because no known transport mechanisms exist, this leakage was attributed to direct membrane damage. In addition, \rm\lbrack Ca\sp{2+}\rbrack\sb{i} and LDH were significantly higher for loading rates above 100 \rm dyne\cdot cm\sp{-2}\cdot s\sp{-1}, whereas quasistatic controls (below 20 \rm dyne\cdot cm\sp{-2}\cdot s\sp{-1}) exhibited no significant increases in either parameter. Since LDH release at 24 hr was elevated for high loading rates, different mechanisms of calcium flux were examined to assess possible pathways which may lead to cell death. Most of the free calcium was found to enter the cytosol from the extracellular space. The presence of extracellular calcium was necessary for cell death. In addition, extracellular glutamate was found to increase significantly 24 hr following high loading rate conditions. The NMDA receptor antagonist MK-801 attenuated injury induced \rm\lbrack Ca\sp{2+}\rbrack\sb{i} increases by 45% and reduced LDH release by 50%. Pretreatment with the calcium channel blocker nifedipine, the glutamate release inhibitor riluzole, and the ganglioside GM1 also significantly reduced \rm\lbrack Ca\sp{2+}\rbrack\sb{i}, but did not affect cell viability. The rate dependent behavior of NTera2 neurons suggests that mechanical loading directly causes changes in membrane permeability which result in calcium influx and membrane depolarization, which trigger cell damaging events. Hindered diffusion of LDH through a porous membrane was analyzed using hydrodynamic theory and then the behavior of calcium peaks and transients was predicted. Assuming short-lived pores formed from high rate deformation, this analysis agreed with the rate dependent behavior seen experimentally and may explain graded responses to injury as a function of mechanical input

Deformation response and cytosolic calcium increases in NTera2 neurons subjected to traumatic levels of hydrodynamic shear stress

A biomechanical method was developed to model traumatic brain injury in vitro by hydrodynamically deforming NTera2 neurons while monitoring intracellular free calcium concentration ( (Ca\sp{2+}\rbrack\sb{\rm i}). Fluid shear stress caused membrane strain rates greater than 1 s\sp{-1}, loading conditions which mimic inertial traumatic loading. Cellular strain, measured by tracking membrane-bound microspheres, ranged from 0 to 52%. \rm\lbrack Ca\sp{2+}\rbrack\sb{i}, assesses by Fura-2, increased with strain and loading rate. Furthermore, \rm\lbrack Ca\sp{2+}\rbrack\sb{i} increases correlated with strain (R\sp2 = 0.75), suggesting a deformation induced injury. Leakage of cytosolic lactate dehydrogenase (LDH) was used to assess cell viability in the first 24 hr following injury. Acute ($\u3c$5 min) LDH release correlated with loading rate (R\sp2 = 0.81) and, because no known transport mechanisms exist, this leakage was attributed to direct membrane damage. In addition, \rm\lbrack Ca\sp{2+}\rbrack\sb{i} and LDH were significantly higher for loading rates above 100 \rm dyne\cdot cm\sp{-2}\cdot s\sp{-1}, whereas quasistatic controls (below 20 \rm dyne\cdot cm\sp{-2}\cdot s\sp{-1}) exhibited no significant increases in either parameter. Since LDH release at 24 hr was elevated for high loading rates, different mechanisms of calcium flux were examined to assess possible pathways which may lead to cell death. Most of the free calcium was found to enter the cytosol from the extracellular space. The presence of extracellular calcium was necessary for cell death. In addition, extracellular glutamate was found to increase significantly 24 hr following high loading rate conditions. The NMDA receptor antagonist MK-801 attenuated injury induced \rm\lbrack Ca\sp{2+}\rbrack\sb{i} increases by 45% and reduced LDH release by 50%. Pretreatment with the calcium channel blocker nifedipine, the glutamate release inhibitor riluzole, and the ganglioside GM1 also significantly reduced \rm\lbrack Ca\sp{2+}\rbrack\sb{i}, but did not affect cell viability. The rate dependent behavior of NTera2 neurons suggests that mechanical loading directly causes changes in membrane permeability which result in calcium influx and membrane depolarization, which trigger cell damaging events. Hindered diffusion of LDH through a porous membrane was analyzed using hydrodynamic theory and then the behavior of calcium peaks and transients was predicted. Assuming short-lived pores formed from high rate deformation, this analysis agreed with the rate dependent behavior seen experimentally and may explain graded responses to injury as a function of mechanical input

PROBLEM-BASED LEARNING IN BIOMEDICAL ENGINEERING CURRICULA

Abstract ⎯ Problem-based Learning (PBL) anchors learning and instruction in concrete problems. We believe that PBL is well suited to educating undergraduate and graduate students within the interdisciplinary field of biomedical engineering (BME). BME draws upon many traditional disciplines to address a range of problems, from biotechnology to clinical medicine. A challenge for BME educators is to balance this broad base of fundamentals with the analytical, in depth problem solving necessary to be successful bioengineers. The ability to adapt, be innovative, and acquire and integrate relevant information is not efficiently learned in a lecture format, but rather in a small group setting that encourages self-directed learning, such as PBL. We have developed a graduate BME program with PBL as one of the pivotal components and are embarking on the introduction of this methodology to undergraduate sections. We have found PBL to be an effective vehicle for instruction, retention of material, and introduction of topics necessary for professional development

Ambient Nanoelectrospray Ionization with In-Line Microdialysis for Spatially Resolved Transient Biochemical Monitoring within Cell Culture Environments

We have developed a new mass spectrometry (MS) based approach for continuous, spatially resolved in vitro biochemical detection and demonstrated its utility in a 3-D cell culture system. Extracellular liquid is passively extracted at a low flow rate (∼10 nL/s) through a small bore silica capillary (ID 50 μm); inline microdialysis (MD) removes ions that would interfere with mass spectrometric analysis, and the sample is ionized by nanoelectrospray ionization (nano-ESI) and mass analyzed in a time-of-flight mass spectrometer. The system successfully detects low-volume, low-concentration releases of a small protein (8 μL of 5 μM cytochrome-c, molecular mass ∼12 kDa) and exhibits ∼1 min temporal resolution. The system also displays sensitivity to probe proximity to the sample release point. Due to the sensitivity of ESI-MS and its ability to simultaneously detect and identify multiple unanticipated biochemicals, this approach shows considerable potential as a biomarker discovery tool

A three-dimensional image processing program for accurate, rapid, and semi-automated segmentation of neuronal somata with dense neurite outgrowth

Three-dimensional (3-D) image analysis techniques provide a powerful means to rapidly and accurately assess complex morphological and functional interactions between neural cells. Current software-based identification methods of neural cells generally fall into two applications: (1) segmentation of cell nuclei in high-density constructs or (2) tracing of cell neurites in single cell investigations. We have developed novel methodologies to permit the systematic identifica-tion of populations of neuronal somata possessing rich morphological detail and dense neurite arborization throughout thick tissue or 3-D in vitro constructs. The image analysis incorporates several novel automated features for the discrimination of neurites and somata by initially classi-fying features in 2-D and merging these classifications into 3-D objects, the 3-D reconstructions automatically identify and adjust for over and under segmentation errors. Additionally, the plat-form provides for software-assisted error corrections to further minimize error. These features attain very accurate cell boundary identifications to handle a wide range of morphological com-plexities. We validated these tools using confocal z-stacks from thick 3-D neural constructs where neuronal somata had varying degrees of neurite arborization and complexity, achieving an accuracy of ≥ 95%. We demonstrated the robustness of these algorithms in a more complex are-na through the automated segmentation of neural cells in ex vivo brain slices. The novel methods surpass previous research improving the robustness and accuracy by: (1) the ability to process neurites and somata, (2) bidirectional segmentation correction, and (3) validation via software-assisted user input. This 3-D image analysis platform provides valuable tools for the unbiased analysis of neural tissue or tissue surrogates within a 3-D context, appropriate for the study of multi-dimensional cell-cell and cell-extracellular matrix interactions