Long-Term Training with a Brain-Machine Interface-Based Gait Protocol Induces Partial Neurological Recovery in Paraplegic Patients.

Brain-machine interfaces (BMIs) provide a new assistive strategy aimed at restoring mobility in severely paralyzed patients. Yet, no study in animals or in human subjects has indicated that long-term BMI training could induce any type of clinical recovery. Eight chronic (3-13 years) spinal cord injury (SCI) paraplegics were subjected to long-term training (12 months) with a multi-stage BMI-based gait neurorehabilitation paradigm aimed at restoring locomotion. This paradigm combined intense immersive virtual reality training, enriched visual-tactile feedback, and walking with two EEG-controlled robotic actuators, including a custom-designed lower limb exoskeleton capable of delivering tactile feedback to subjects. Following 12 months of training with this paradigm, all eight patients experienced neurological improvements in somatic sensation (pain localization, fine/crude touch, and proprioceptive sensing) in multiple dermatomes. Patients also regained voluntary motor control in key muscles below the SCI level, as measured by EMGs, resulting in marked improvement in their walking index. As a result, 50% of these patients were upgraded to an incomplete paraplegia classification. Neurological recovery was paralleled by the reemergence of lower limb motor imagery at cortical level. We hypothesize that this unprecedented neurological recovery results from both cortical and spinal cord plasticity triggered by long-term BMI usage.

In vivo and ex vivo imaging of amyloid-ß cascade aggregates with a Pronucleon™ peptide.

Accumulation of amyloid-ß (Aß) cascade aggregates is considered a hallmark of Alzheimer's disease (AD). Current dogma holds that the appearance of Aß oligomers and larger aggregates occur many years prior to plaque formation associated with the advanced and irreparable neurocognitive decline characteristic of AD. This premise is the impetus to identify these Aß precursor structures prior to advanced plaque development. The Pronucleon™ technology platform is comprised of a novel series of engineered peptides that provide a unique readout when associated with beta-rich fiber and oligomeric Aß. This technology has been applied to Ex Vivo tissue sections and In Vivo mouse models of AD to determine the potential utility of these synthetic peptides as potential imaging agents. In Ex Vivo studies, the Pronucleon™ peptide binds plaque like structures in brain sections obtained from transgenic mice overexpressing hAPP with both the human Swedish and London Aß mutations. In Vivo, Pronucleon™ peptide administered peripherally can localize to the brain and label plaques throughout the brain in transgenic mice. Taken together, the data suggest that Pronucleon™ could provide a new imaging tool for Aß cascade elements that precede advanced plaque and fibril formation, thereby advancing early diagnosis and treatment opportunities.

Structure-selective anisotropy assay for amyloid Beta oligomers.

Amyloid ß (Abeta) peptides in their oligomeric form have been proposed as major toxic species in Alzheimer's disease (AD). There are a limited number of anti-Abeta antibodies specific to oligomeric forms of Abeta compared to the monomeric form, and accurate measurement of oligomeric forms in biological samples, cerebrospinal fluid (CSF), or brain extracts remains challenging. We introduce an oligomer-specific (in preference to monomers or fibrils) fluorescence assay based on a conformationally sensitive bis-pyrene-labeled peptide that contains amino acid residues 16-35 of the human amyloid beta protein (pronucleon peptide, PP). This peptide exhibits a shift in fluorescence emission from pyrene excimer to pyrene monomer emission resulting from a conformational change. Specific binding of PP to oligomeric forms of Abeta can be monitored in solution by a change in fluorescence spectrum as well as a change in pyrene monomer fluorescence anisotropy (or polarization). The mechanism of binding and its relation to anisotropy and fluorescence lifetime changes are discussed. The development of a simple, rapid, anisotropy assay for measurement of Abeta oligomers is important for further study of the oligomers' role in AD, and specific detection of oligomers in biological samples, such as cerebrospinal fluid.

Detection of misfolded prion protein in blood with conformationally sensitive peptides.

BACKGROUND: The long-standing goal of a preclinical diagnostic test for transmissible spongiform encephalopathy (TSE) has recently become urgent because of the discovery that humans with variant Creutzfeldt-Jakob disease can transmit disease via blood transfusions. STUDY DESIGN AND METHODS: The misfolded protein diagnostic (MPD) assay employs a pyrene-labeled palindromic sequence of prion peptides that undergoes a cascade of coil to beta-sheet conversion in the presence of the misfolded prion protein (PrP(TSE)). The ability of the assay to detect PrP(TSE) in brain, serum, and plasma was tested. The basic protocol involved a several-hour incubation of 200-microL sample volumes with the peptide reagent in 96-well plates, after which fluorescence was monitored by a fluorescence plate reader with an excitation wavelength of 350 nm and emission scanning wavelength range of 365 to 600 nm. RESULTS: Target specificity for PrP(TSE) was documented by correlation of assay signal with Western blot signals in brain tissue from TSE-infected, normal, and knockout mice and negative assay signals by use of reagents with different peptide sequences. When applied to plasma or serum, the assay discriminated between samples from a variety of experimental and natural TSE infections compared to uninfected controls, with a sensitivity threshold of approximately 1 infectious dose per mL in pooled plasma from TSE-infected mice. CONCLUSIONS: The MPD assay is a sensitive and specific test for the detection of PrP(TSE) that may be useful in both preclinical and clinical diagnosis of TSE diseases of animals and humans.

Trehalose lyophilized platelets for wound healing.

Fresh platelet preparations are utilized to treat a wide variety of wounds, although storage limitations and mixed results have hampered their clinical use. We hypothesized that concentrated lyophilized and reconstituted platelet preparations, preserved with trehalose, maintain and possibly enhance fresh platelets' ability to improve wound healing. We studied the ability of a single dose of trehalose lyophilized and reconstituted platelets to enhance wound healing when topically applied on full-thickness wounds in the genetically diabetic mouse. We compared these results with the application of multiple doses of fresh platelet preparations and trehalose lyophilized and reconstituted platelets as well as multiple doses of vascular endothelial growth factor (VEGF) and wounds left untreated. Trehalose lyophilized and reconstituted platelets, in single and multiple applications, multiple applications of fresh platelets and multiple applications of VEGF increased granulation tissue deposition, vascularity, and proliferation when compared with untreated wounds, as assessed by histology and immunohistochemistry. Wounds treated with multiple doses of VEGF and a single dose of freeze-dried platelets reached 90% closure faster than wounds left untreated. A single administration of trehalose lyophilized and reconstituted platelet preparations enhanced diabetic wound healing, therefore representing a promising strategy for the treatment of nonhealing wounds.

Wound-healing properties of trehalose-stabilized freeze-dried outdated platelets.

BACKGROUND: The wound-healing applications of platelet (PLT)-derived cytokines, proteins, and membranes is accepted but continues to be investigated. In this study, it is demonstrated that stabilized freeze-dried PLTs prepared from outdated PLTs (FDPOs) accelerate wound healing and form tube structures as well as stabilized indated freeze-dried PLTs (FDPIs) and room-temperature fresh PLTs (RT-PLTs). STUDY DESIGN AND METHODS: Experiments were designed to compare in vitro and in vivo wound-healing properties of FDPI, FDPO, and RT-PLT preparations. The concentration of PLT-derived growth factor (PDGF)-betabeta and transforming growth factor (TGF)-beta1 was determined, and the abilities of FDPIs, FDPOs and RT-PLTs to induce endothelial cell proliferation and promote endothelial cell tube formation (cells formed solid spouts connecting neighboring cells to form tube structures) were observed. Wound-healing characteristics were measured by surgically inducing 1-cm(2), full-thickness wounds on db/db mice (n = 10 per group). The wounds were treated with single or multiple doses of FDPIs and FDPOs. Wound closure rate was determined, and histology samples were evaluated for cellular makeup. RESULTS: FDPOs retained the same levels of PDGF-betabeta and TGF-beta1 and were able to promote endothelial cell proliferation and tube formation in vitro as well as FDPIs or RT-PLTs. Multiple applications of FDPO accelerated wound closure and enhanced reepithelialization when compared to untreated wounds in db/db mice. CONCLUSION: FDPOs enhanced wound healing in db/db mice as well as FDPIs and RT-PLTs. Wound closure was obtained 6 days earlier than untreated wounds and histologic examination revealed reduced granulation and increased cellular angiogenesis.