Engineering angiogenesis following spinal cord injury: a coculture of neural progenitor and endothelial cells in a degradable polymer implant leads to an increase in vessel density and formation of the blood–spinal cord barrier

Angiogenesis precedes recovery following spinal cord injury and its extent correlates with neural regeneration, suggesting that angiogenesis may play a role in repair. An important precondition for studying the role of angiogenesis is the ability to induce it in a controlled manner. Previously, we showed that a coculture of endothelial cells (ECs) and neural progenitor cells (NPCs) promoted the formation of stable tubes in vitro and stable, functional vascular networks in vivo in a subcutaneous model. We sought to test whether a similar coculture would lead to the formation of stable functional vessels in the spinal cord following injury. We created microvascular networks in a biodegradable two-component implant system and tested the ability of the coculture or controls (lesion control, implant alone, implant + ECs or implant + NPCs) to promote angiogenesis in a rat hemisection model of spinal cord injury. The coculture implant led to a fourfold increase in functional vessels compared with the lesion control, implant alone or implant + NPCs groups and a twofold increase in functional vessels over the implant + ECs group. Furthermore, half of the vessels in the coculture implant exhibited positive staining for the endothelial barrier antigen, a marker for the formation of the blood–spinal cord barrier. No other groups have shown positive staining for the blood–spinal cord barrier in the injury epicenter. This work provides a novel method to induce angiogenesis following spinal cord injury and a foundation for studying its role in repair.     

Macrophage activation and Fcgamma receptor-mediated signaling do not require expression of the SLP-76 and SLP-65 adaptors

The Src-homology 2 domain-containing, leukocyte-specific phosphoprotein of 76 kDa (SLP-76) is a hematopoietic adaptor that plays a central role during immunoreceptor-mediated activation of T lymphocytes and mast cells and collagen receptor-induced activation of platelets. Despite similar levels of expression in macrophages, SLP-76 is not required for Fc receptor for immunoglobulin G (IgG; FcgammaR)-mediated activation. We hypothesized that the related adaptor SLP-65, which is also expressed in macrophages, may compensate for the loss of SLP-76 during FcgammaR-mediated signaling and functional events. To address this hypothesis, we examined bone marrow-derived macrophages (BMM) from wild-type (WT) mice or mice lacking both of these adaptors. Contrary to our expectations, SLP-76(-/-) SLP-65(-/-) BMM demonstrated normal FcgammaR-mediated activation, including internalization of Ig-coated sheep red blood cells and production of reactive oxygen intermediates. FcgammaR-induced biochemical events were normal in SLP-76(-/-) SLP-65(-/-) BMM, including phosphorylation of phospholipase C and the extracellular signaling-regulated kinases 1 and 2. To determine whether macrophages functioned normally in vivo, we infected WT and SLP-76(-/-) SLP-65(-/-) mice with sublethal doses of Listeria monocytogenes (LM), a bacterium against which the initial host defense is provided by activated macrophages. WT and SLP-76(-/-) SLP-65(-/-) mice survived acute, low-dose infection and showed no difference in the number of liver or spleen LM colony-forming units, a measure of the total body burden of this organism. Taken together, these data suggest that neither SLP-76 nor SLP-65 is required during FcgammaR-dependent signaling and functional events in macrophages.     

Real-time imaging of Rab3a and Rab5a reveals differential roles in presynaptic function

We investigated the roles of two Rab-family proteins, Rab3a and Rab5a, in hippocampal synaptic transmission using real-time fluorescence imaging. During synaptic activity, Rab3a dissociated from synaptic vesicles and dispersed into neighbouring axonal regions. Dispersion required calcium-dependent exocytosis and was complete before the entire vesicle pool turned over. In contrast, even prolonged synaptic activity produced limited dispersion of Rab5a. A GTPase-deficient mutant, Rab3a (Q81L), dispersed more slowly than wild-type Rab3a, and decreased the rate of exocytosis and the size of the recycling pool of vesicles. While overexpression of Rab3a did not affect vesicle recycling, overexpression of Rab5a reduced the recycling pool size by 50%. We propose that while Rab3a preferentially associates with recycling synaptic vesicles and modulates their trafficking, Rab5a is largely excluded from recycling vesicles.     

Spontaneous waves in the dentate gyrus of slices from the ventral hippocampus

Spontaneous negative-going potentials occurring at an average frequency of 0.7 Hz were recorded from the dentate gyrus of slices prepared from the temporal hippocampus of young adult rats. These events (here termed "dentate waves") in several respects resembled the dentate spikes described for freely moving rats during immobile behaviors and slow-wave sleep. Action potentials were observed on the descending portion of the in vitro waves and, as expected from this, whole cell recordings established that the waves were composed of depolarizing currents. Dentate waves appeared to be locally generated within the granule cell layer and were greatly reduced by antagonists of AMPA-type glutamate receptors or by lesions to the entorhinal cortex. Simultaneous recordings indicated that the waves were often synchronized in the inner and outer blades of the dentate gyrus. Knife cuts through the perforant path and the commissural/associational system did not eliminate synchronization, leaving electrotonic propagation via gap junctions as its probable cause. In accord with this, cuts that separated the two blades of the dentate eliminated synchronization between them, and a compound that inhibits gap junctions reduced wave activity. Dentate waves were regularly accompanied by sharp waves in field CA3 and were reduced in size by the acetylcholinesterase inhibitor, physostigmine. It is hypothesized that dentate waves occur when spontaneous glutamate release from dentate afferents produces action potentials in neighboring granule cells that then summate electrotonically into a population event; once initiated, the waves propagate, again electrotonically, and thereby engage a significant portion of the granule cell population.     

Effects of a yearlong moderate-intensity exercise and a stretching intervention on sleep quality in postmenopausal women

STUDY OBJECTIVES: To examine the effects of a moderate-intensity exercise or stretching intervention and changes in fitness, body mass index, or time spent outdoors on self-reported sleep quality and to examine the relationship between the amount and timing of exercise and sleep quality. DESIGN: A randomized intervention trial. SETTING: A cancer research center in Seattle, Washington. PARTICIPANTS: Postmenopausal, overweight or obese, sedentary women not taking hormone replacement therapy, aged 50 to 75 years, and recruited from the Seattle metropolitan area. INTERVENTIONS: A yearlong moderate-intensity exercise (n=87) and a low-intensity stretching (n=86) program. MEASUREMENTS AND RESULTS: Among morning exercisers, those who exercised at least 225 minutes per week had less trouble falling asleep (odds ratio [OR]: 0.3, P < or = .05) compared with those who exercised less than 180 minutes per week. However, among evening exercisers, those who exercised at least 225 minutes per week had more trouble falling asleep (OR: 3.3, P < or = .05) compared to those who exercised less than 180 minutes per week. Stretchers were less likely to use sleep medication (OR = 0.4, P < or = .05) and have trouble falling asleep (OR: 0.7, P < or = .10) during the intervention period compared with baseline. A greater than 10% versus a 1% or less increase in maximum O2 consumption over the year was associated with longer sleep duration (P < or = .05), less frequently falling asleep during quiet activities (P < or = .05), and less use of sleep medication (P < or = .05). Reductions in body mass index and increases in time spent outdoors had inconsistent effects on sleep quality. CONCLUSIONS: Both stretching and exercise interventions may improve sleep quality in sedentary, overweight, postmenopausal women. Increased fitness was associated with improvements in sleep. However, the effect of moderate-intensity exercise may depend on the amount of exercise and time of day it is performed.     

Nutrient signalling in the regulation of human muscle protein synthesis

The mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) are important nutrient- and energy-sensing and signalling proteins in skeletal muscle. AMPK activation decreases muscle protein synthesis by inhibiting mTOR signalling to regulatory proteins associated with translation initiation and elongation. On the other hand, essential amino acids (leucine in particular) and insulin stimulate mTOR signalling and protein synthesis. We hypothesized that anabolic nutrients would be sensed by both AMPK and mTOR, resulting in an acute and potent stimulation of human skeletal muscle protein synthesis via enhanced translation initiation and elongation.
We measured muscle protein synthesis and mTOR-associated upstream and downstream signalling proteins in young male subjects ( n = 14) using stable isotopic and immunoblotting techniques. Following a first muscle biopsy, subjects in the 'Nutrition' group ingested a leucine-enriched essential amino acid–carbohydrate mixture (EAC). Subjects in the Control group did not consume nutrients. A second biopsy was obtained 1 h later. Ingestion of EAC significantly increased muscle protein synthesis, modestly reduced AMPK phosphorylation, and increased Akt/PKB (protein kinase B) and mTOR phosphorylation ( P < 0.05). mTOR signalling to its downstream effectors (S6 kinase 1 (S6K1) and 4E-binding protein 1 (4E-BP1) phosphorylation status) was also increased ( P < 0.05). In addition, eukaryotic elongation factor 2 (eEF2) phosphorylation was significantly reduced ( P < 0.05). Protein synthesis and cell signalling (phosphorylation status) was unchanged in the control group ( P > 0.05).
We conclude that anabolic nutrients alter the phosphorylation status of both AMPK- and mTOR-associated signalling proteins in human muscle, in association with an increase in protein synthesis not only via enhanced translation initiation but also through signalling promoting translation elongation.     

Co-detection and discrimination of six human herpesviruses by multiplex PCR-ELAHA.

BACKGROUND: Herpesviruses are a significant cause of human morbidity. Traditional approaches to the identification of these viruses require infectious or at least antigenic virus. Multiplex PCR (mPCR) is capable of simultaneously amplifying a range of targets from a single preparation of nucleic acids and when combined with a suitable detection assay, it is capable of discriminating each of the amplicons. OBJECTIVES: Several methods have been described in the literature, however, they lack one or more significant design features required to suitably control a routinely applied nucleic acid amplification assay. We aimed to design a multiplex herpesvirus PCR that could co-amplify eight human herpesvirus targets plus an internal control (IC) molecule in a single tube. STUDY DESIGN: Primers were designed to target the DNA polymerase genes of each of the human herpesviruses. Synthetic controls were developed to act as templates for the evaluation of assay sensitivity and specificity and for development of an in-house competitive quantitative PCR. Amplicon was discriminated using a simplified enzyme linked amplicon hybridisation assay (ELAHA). RESULTS AND CONCLUSIONS: For routine diagnostic use we reduced the number of herpesviral targets from 8 to 6 in order to maintain adequate clinical sensitivity. The ELAHA proved more sensitive than agarose gel electrophoresis. Additionally, 36 cytomegalovirus positive patients were examined with an in-house quantitative PCR-ELAHA which was developed to confirm that that the mPCR's co-detection limit of 10(2) copy of synthetic template per millilitre was relevant for use in detecting virus from clinical samples. The mPCR-ELAHA was then applied to the screening of 174 patient specimens resulting in a specificity of 98% and a sensitivity of 93%. This preliminary study demonstrated that the mPCR-ELAHA was a complete approach to the detection of herpesviruses from a range of clinical samples and disease states.