A model for overcoming challenges in academic pediatric medical device innovation.

Technological advancements in medical devices developed for adults far outpace the development of technologies designed for pediatric patients in the USA and other countries. This technology lag was previously reflected in a lack of pediatric-specific innovation within our academic institution. To address the institutional deficit of device innovation around pediatric patients, we formed unique partnerships both within our university and extending to the medical device industry, and developed novel programmatic approaches. The Pediatric Device Innovation Consortium (PDIC) bridges the medical device community and the University of Minnesota. Since 2014, the PDIC has supported 22 pediatric medical technology innovation projects, provided funds totaling more than $500,000, licensed two technologies, and advanced two technologies to patient use. Here, we describe the PDIC model and method, the PDIC approach to common challenges that arise in the development of small-market medical technologies at an academic institution, and iterations to our collaborative, multidisciplinary approach that have matured throughout our experience. The PDIC model continues to evolve to reflect the special needs of innovation for smaller markets and the unique role of clinician innovators. Our approach serves as a successful model for other institutions interested in creating support mechanisms for pediatric or small-market technology development.

Estradiol Facilitation of Cocaine Self-Administration in Female Rats Requires Activation of mGluR5.

In comparison to men, women initiate drug use at earlier ages and progress from initial use to addiction more rapidly. This heightened intake and vulnerability to drugs of abuse is regulated in part by estradiol, although the signaling mechanisms by which this occurs are not well understood. Recent findings indicate that within the nucleus accumbens core, estradiol induces structural plasticity via membrane-localized estrogen receptor α, functionally coupled to metabotropic glutamate receptor subtype 5 (mGluR5). Hence, we sought to determine whether mGluR5 activation was essential for estradiol-mediated enhancement of cocaine self-administration. Ovariectomized (OVX) female rats were allowed to freely self-administer cocaine under extended access conditions (6 h/d) for 10 consecutive days. The mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) or vehicle was administered before estradiol (or oil), on a 2 d on/2 d off schedule throughout the extended access period. MPEP treatment prevented the estradiol-dependent enhancement of cocaine self-administration in OVX females. In a separate experiment, potentiation of mGluR5 function with the positive allosteric modulator 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (in the absence of estradiol treatment) failed to increase cocaine self-administration. These data suggest that mGluR5 activation is necessary for estradiol-mediated enhancement of responses to cocaine, but that direct mGluR5 activation is insufficient to mimic the female response to estradiol. Building on previous studies in male animals, these findings further highlight the therapeutic potential of mGluR5 antagonism in the treatment of addiction and suggest that there may be added therapeutic benefit in females.

Estradiol impacts the endocannabinoid system in female rats to influence behavioral and structural responses to cocaine.

Compared with men, women show enhanced responses to drugs of abuse, and consequently are thought to be more vulnerable to addiction. The ovarian hormone estradiol has emerged as a key facilitator in the heightened development of addiction in females. These actions of estradiol appear mediated by estrogen receptor (ER) activation of metabotropic glutamate receptor type 5 (mGluR5). However, the downstream effectors of this ER/mGluR5 signaling pathway are unknown. Here we investigate whether cannabinoid 1 receptor (CB1R) activation is a part of the mechanism whereby estradiol influences behavioral and synaptic correlates of addiction. Following repeated cocaine administration, estradiol-treated ovariectomized rats exhibited both sensitized locomotor responses and decreases in the dendritic spine density of nucleus accumbens core medium-spiny neurons in comparison to oil-treated controls. Both effects of estradiol were blocked by AM251, a CB1R inverse agonist. These results indicate that part of the signaling mechanism through which estradiol impacts behavioral and synaptic correlates of addiction in female rats requires activation of CB1Rs.

Impact of immersion oils and mounting media on the confocal imaging of dendritic spines.

BACKGROUND: Structural plasticity, such as changes in dendritic spine morphology and density, reflect changes in synaptic connectivity and circuitry. Procedural variables used in different methods for labeling dendritic spines have been quantitatively evaluated for their impact on the ability to resolve individual spines in confocal microscopic analyses. In contrast, there have been discussions, though no quantitative analyses, of the potential effects of choosing specific mounting media and immersion oils on dendritic spine resolution. NEW METHOD: Here we provide quantitative data measuring the impact of these variables on resolving dendritic spines in 3D confocal analyses. Medium spiny neurons from the rat striatum and nucleus accumbens are used as examples. RESULTS: Both choice of mounting media and immersion oil affected the visualization of dendritic spines, with choosing the appropriate immersion oil as being more imperative. These biologic data are supported by quantitative measures of the 3D diffraction pattern (i.e. point spread function) of a point source of light under the same mounting medium and immersion oil combinations. COMPARISON WITH EXISTING METHOD: Although not a new method, this manuscript provides quantitative data demonstrating that different mounting media and immersion oils can impact the ability to resolve dendritic spines. These findings highlight the importance of reporting which mounting medium and immersion oil are used in preparations for confocal analyses, especially when comparing published results from different laboratories. CONCLUSION: Collectively, these data suggest that choosing the appropriate immersion oil and mounting media is critical for obtaining the best resolution, and consequently more accurate measures of dendritic spine densities.

Estradiol mediates dendritic spine plasticity in the nucleus accumbens core through activation of mGluR5.

Accumulating evidence from human and rodent studies suggests that females are more sensitive to the motivating and rewarding properties of drugs of abuse. Numerous reports implicate estradiol in enhancing drug-related responses in females, yet the neurobiological mechanisms underlying this effect of estradiol are unknown. Because dendritic spine plasticity in the nucleus accumbens (NAc) is linked to the addictive effects of drugs, we examined the influence of estradiol on dendritic spines in this region. Previously our laboratory demonstrated that in female medium spiny neurons, estradiol activates metabotropic glutamate receptor subtype five (mGluR5), a G protein-coupled receptor already implicated in the etiology of drug addiction. Thus, we sought to determine whether mGluR5 is a part of the mechanism by which estradiol affects dendritic spine density in the NAc. To test this hypothesis, ovariectomized female rats were treated with the mGluR5 antagonist, MPEP, or vehicle prior to estradiol (or oil) treatment and 24 h later dendritic spine density was evaluated by DiI labeling and confocal microscopy. We found that estradiol decreased dendritic spine density in the NAc core and that pretreatment with MPEP blocked this effect. In contrast, MPEP had no effect on dendritic spine density in the NAc shell or CA1 region of the hippocampus, two regions in which estradiol increased the density of dendritic spines. As dendritic spine plasticity in the NAc core has behavioral consequences for drug addiction, these data provide a clue as to how estradiol acts in females to enhance behavioral responses to drugs of abuse.

Estradiol facilitation of cocaine-induced locomotor sensitization in female rats requires activation of mGluR5.

In comparison to men, women exhibit enhanced responsiveness to the stimulating and addictive properties of cocaine. A growing body of evidence implicates the steroid hormone estradiol in mediating this sex difference, yet the mechanisms underlying estradiol enhancement of behavioral responses to cocaine in females are not known. Recently, we have found that estrogen receptor alpha (ERα) functionally couples with the metabotropic glutamate receptor 5 (mGluR5) to mediate the effects of estradiol on both cellular activation as well as dendritic spine plasticity in brain regions involved in cocaine-induced behavioral sensitization. Thus, we sought to determine whether mGluR5 activation is required for the facilitative effects of estradiol on locomotor responses to cocaine. To test this hypothesis, ovariectomized (OVX) female rats were tested for locomotor activity on the first and fifth days of daily systemic injections of cocaine. For the 2 days prior to each locomotor test, animals were injected with the mGluR5 antagonist MPEP (or vehicle) and estradiol (or oil). MPEP treatment blocked the facilitative effects of estradiol on cocaine-induced locomotor sensitization, without affecting acute responses to cocaine or the inhibitory actions of estradiol on weight gain. Considered together, these data indicate that mGluR5 activation is critical for the actions of estradiol on cocaine-induced behavioral sensitization.

Palmitoylation of estrogen receptors is essential for neuronal membrane signaling.

In addition to activating nuclear estrogen receptor signaling, 17ß-estradiol can also regulate neuronal function via surface membrane receptors. In various brain regions, these actions are mediated by the direct association of estrogen receptors (ERs) activating metabotropic glutamate receptors (mGluRs). These ER/mGluR signaling partners are organized into discrete functional microdomains via caveolin proteins. A central question that remains concerns the underlying mechanism by which these subpopulations of ERs are targeted to the surface membrane. One candidate mechanism is S-palmitoylation, a posttranscriptional modification that affects the subcellular distribution and function of the modified protein, including promoting localization to membranes. Here we test for the role of palmitoylation and the necessity of specific palmitoylacyltransferase proteins in neuronal membrane ER action. In hippocampal neurons, pharmacological inhibition of palmitoylation eliminated 17ß-estradiol-mediated phosphorylation of cAMP response element-binding protein, a process dependent on surface membrane ERs. In addition, mutation of the palmitoylation site on estrogen receptor (ER) α blocks ERα-mediated cAMP response element-binding protein phosphorylation. Similar results were obtained after mutation of the palmitoylation site on ERß. Importantly, mutation of either ERα or ERß did not affect the ability of the reciprocal ER to signal at the membrane. In contrast, membrane ERα and ERß signaling were both dependent on the expression of the palmitoylacyltransferase proteins DHHC-7 and DHHC-21. Neither mGluR activity nor caveolin or ER expression was affected by knockdown of DHHC-7 and DHHC-21. These data collectively suggest discrete mechanisms that regulate specific isoform or global membrane ER signaling in neurons separate from mGluR activity or nuclear ER function.

Adolescent changes in dopamine D1 receptor expression in orbitofrontal cortex and piriform cortex accompany an associative learning deficit.

The orbitofrontal cortex (OFC) and piriform cortex are involved in encoding the predictive value of olfactory stimuli in rats, and neural responses to olfactory stimuli in these areas change as associations are learned. This experience-dependent plasticity mirrors task-related changes previously observed in mesocortical dopamine neurons, which have been implicated in learning the predictive value of cues. Although forms of associative learning can be found at all ages, cortical dopamine projections do not mature until after postnatal day 35 in the rat. We hypothesized that these changes in dopamine circuitry during the juvenile and adolescent periods would result in age-dependent differences in learning the predictive value of environmental cues. Using an odor-guided associative learning task, we found that adolescent rats learn the association between an odor and a palatable reward significantly more slowly than either juvenile or adult rats. Further, adolescent rats displayed greater distractibility during the task than either juvenile or adult rats. Using real-time quantitative PCR and immunohistochemical methods, we observed that the behavioral deficit in adolescence coincides with a significant increase in D1 dopamine receptor expression compared to juvenile rats in both the OFC and piriform cortex. Further, we found that both the slower learning and increased distractibility exhibited in adolescence could be alleviated by experience with the association task as a juvenile, or by an acute administration of a low dose of either the dopamine D1 receptor agonist SKF-38393 or the D2 receptor antagonist eticlopride. These results suggest that dopaminergic modulation of cortical function may be important for learning the predictive value of environmental stimuli, and that developmental changes in cortical dopaminergic circuitry may underlie age-related differences in associative learning.

SDF-1α secreted by human CD133-derived multipotent stromal cells promotes neural progenitor cell survival through CXCR7.

We recently reported that concentrated conditioned medium (CdM) from human CD133-derived bone marrow progenitor cells (CD133 CdM) was neuroprotective after stroke. Here we identify stromal-derived factor 1 alpha (SDF-1) as a potential neuroprotective candidate in CD133 CdM by interrogating the transcriptional responses of CD133-derived multipotent stromal cells (CD133dMSCs) after cell injection into the ischemic brain. Human SDF-1 mRNA was upregulated 79-fold by CD133dMSCs when injected into the stroke peri-infarct area compared with cells injected into the uninjured parenchyma of sham-operated animals. In cell protection assays, we replaced the typical growth medium in mouse neural progenitor cell (mNPC) cultures with serum-free CD133 CdM immediately before exposure to hypoxia (1% oxygen) for 48 h. CD133 CdM significantly increased the survival of mNPCs during hypoxia exposure and growth factor withdrawal. To determine whether MSC-secreted SDF-1 influenced mNPC survival, we used lentiviral short hairpin RNA against SDF1 (shSDF-1) to knockdown SDF-1 expression in CD133dMSCs. The CdM generated from shSDF-1-treated cells had a 94% decrease in secreted SDF-1 and was significantly less protective for mNPCs when compared with control CdM from CD133dMSCs transduced with scrambled short hairpin RNA. Pharmacological inhibition of the 2 known SDF-1 receptors, CXCR4 and CXCR7, revealed that only CXCR7 activity was functionally linked to survival signaling in mNPCs during hypoxia exposure. Treatment of mNPCs with CD133 CdM and CXCR7 inhibitor decreased mNPC viability by 36.5% ± 12.8% and decreased cell number by 21% ± 6.7% compared with dimethyl sulfoxide treated controls. These data indicate that SDF-1 is a key neuroprotective cytokine secreted by CD133dMSCs that protects mNPCs through CXCR7.

Isolation of locally derived stem/progenitor cells from the peri-infarct area that do not migrate from the lateral ventricle after cortical stroke.

BACKGROUND AND PURPOSE: Neurogenesis can arise from neural stem/progenitor cells of the subventricular zone after strokes involving both the cortex and striatum. However, it is controversial whether all types of stroke and strokes of different sizes activate neurogenesis from the subventricular zone niche. In contrast with cortical/striatal strokes, repair and remodeling after mild cortical strokes may involve to a greater extent local cortical stem/progenitor cells and cells from nonneurogenic niches. METHODS: We compared stem/progenitor cell responses after focal cortical strokes produced by distal middle cerebral artery occlusion and cortical/striatal strokes produced by the intraluminal suture model. To label migrating neuroblasts from the subventricular zone, we injected DiI to the lateral ventricle after distal middle cerebral artery occlusion. By immunohistochemistry, we characterized cells expressing stem/progenitor cell markers in the peri-infarct area. We isolated cortical stem/progenitor cells from the peri-infarct area after distal middle cerebral artery occlusion and assayed their self-renewal and differentiation capacity. RESULTS: In contrast with cortical/striatal strokes, focal cortical strokes did not induce neuroblast migration from the subventricular zone to the infarct zone after distal middle cerebral artery occlusion. By immunohistochemistry, we observed subpopulations of reactive astrocytes in the peri-infarct area that coexpressed radial glial cell markers such as Sox2, Nestin, and RC2. Clonal neural spheres isolated from the peri-infarct area after distal middle cerebral artery occlusion differentiated into neurons, astrocytes, oligodendrocytes, and smooth muscle cells. Notably, neural spheres isolated from the peri-infarct area also expressed RC2 before differentiation. CONCLUSIONS: Mild cortical strokes that do not penetrate the striatum activate local cortical stem/progenitor cells but do not induce neuroblast migration from the subventricular zone niche.