Basic control of reperfusion effectively protects against reperfusion injury in a realistic rodent model of acute limb ischemia

BACKGROUND: Reperfusion injury is insufficiently addressed in current clinical management of acute limb ischemia. Controlled reperfusion carries an enormous clinical potential and was tested in a new reality-driven rodent model. METHODS AND RESULTS: Acute hind-limb ischemia was induced in Wistar rats and maintained for 4 hours. Unlike previous tourniquets models, femoral vessels were surgically prepared to facilitate controlled reperfusion and to prevent venous stasis. Rats were randomized into an experimental group (n=7), in which limbs were selectively perfused with a cooled isotone heparin solution at a limited flow rate before blood flow was restored, and a conventional group (n=7; uncontrolled blood reperfusion). Rats were killed 4 hours after blood reperfusion. Nonischemic limbs served as controls. Ischemia/reperfusion injury was significant in both groups; total wet-to-dry ratio was 159+/-44% of normal (P=0.016), whereas muscle viability and contraction force were reduced to 65+/-13% (P=0.016) and 45+/-34% (P=0.045), respectively. Controlled reperfusion, however, attenuated reperfusion injury significantly. Tissue edema was less pronounced (132+/-16% versus 185+/-42%; P=0.011) and muscle viability (74+/-11% versus 57+/-9%; P=0.004) and contraction force (68+/-40% versus 26+/-7%; P=0.045) were better preserved than after uncontrolled reperfusion. Moreover, subsequent blood circulation as assessed by laser Doppler recovered completely after controlled reperfusion but stayed durably impaired after uncontrolled reperfusion (P=0.027). CONCLUSIONS: Reperfusion injury was significantly alleviated by basic modifications of the initial reperfusion period in a new in vivo model of acute limb ischemia. With this model, systematic optimizations of according protocols may eventually translate into improved clinical management of acute limb ischemia.

Outcome evaluation of early placed maxillary anterior single-tooth implants using objective esthetic criteria: a cross-sectional, retrospective study in 45 patients with a 2- to 4-year follow-up using pink and white esthetic scores

BACKGROUND: To validate the concept of early implant placement for use in the esthetically sensitive anterior maxilla, clinical trials should ideally include objective esthetic criteria when assessing outcome parameters. METHODS: In this cross-sectional, retrospective 2- to 4-year study involving 45 patients treated with maxillary anterior single-tooth implants according to the concept of early implant placement, a novel comprehensive index, comprising pink esthetic score and white esthetic score (PES/WES; the highest possible combined score is 20), was applied for the objective esthetic outcome assessment of anterior single-tooth implants. RESULTS: All 45 anterior maxillary single-tooth implants fulfilled strict success criteria for dental implants with regard to osseointegration, including the absence of peri-implant radiolucency, implant mobility, suppuration, and pain. The mean total PES/WES was 14.7 +/- 1.18 (range: 11 to 18). The mean total PES of 7.8 +/- 0.88 (range: 6 to 9) documents favorable overall peri-implant soft tissue conditions. The two PES variables facial mucosa curvature (1.9 +/- 0.29) and facial mucosa level (1.8 +/- 0.42) had the highest mean values, whereas the combination variable root convexity/soft tissue color and texture (1.2 +/- 0.53) proved to be the most difficult to fully satisfy. Mean scores were 1.6 +/- 0.5 for the mesial papilla and 1.3 +/- 0.5 for the distal papilla. A mean value of 6.9 +/- 1.47 (range: 4 to 10) was calculated for WES. CONCLUSIONS: This study demonstrated that anterior maxillary single-tooth replacement, according to the concept of early implant placement, is a successful and predictable treatment modality, in general, and from an esthetic point of view, in particular. The suitability of the PES/WES index for the objective outcome assessment of the esthetic dimension of anterior single-tooth implants was confirmed. However, prospective clinical trials are needed to further validate and refine this index.

Chronic critical limb ischemia: European experiences

Chronic critical limb ischemia still poses a substantial threat to both limb and life of the affected patients since these patients suffer typically also from associated cardiac and cerebrovascular disease and other severe comorbidities. Due to improved secondary prevention strategies and dedicated technical innovation, however, clinical outcomes have improved in the recent years. Purpose of this article is to provide a balanced discussion of contemporary treatment concepts for patients with critical limb ischemia with a focus on arterial revascularization.

Call for a reference model of chronic hind limb ischemia to investigate therapeutic angiogenesis

A large number of studies utilize animal models to investigate therapeutic angiogenesis. However, the lack of a standardized experimental model leaves the comparison of different studies problematic. To establish a reference model of prolonged moderate tissue ischemia, we created unilateral hind limb ischemia in athymic rnu-rats by surgical excision of the femoral vessels. Blood flow of the limb was monitored for 60 days by laser Doppler imaging. Following a short postoperative period of substantially depressed perfusion, the animals showed a status of moderate hind limb ischemia from day 14 onwards. Thereafter, the perfusion remained at a constant level (55.5% of normal value) until the end of the observation period. Histopathological assessment of the ischemic musculature on postoperative days 28 and 60 showed essentially no inflammatory cell infiltrate or fibrosis. However, the mitochondrial activity and capillary-to-fiber ratio of the muscular tissue was reduced to 52.7% of normal, presenting with a significant weakness of the ischemic limb evidenced by a progressive decline in performance. Intramuscular injection of culture-expanded human endothelial progenitor cells (EPC) resulted in a significant increase in blood flow (82.0+/-3.5% of normal), capillary density (1.60+/-0.08/muscle fiber) and smooth muscle covered arterioles (8.0+/-0.6/high power field) in the ischemic hind limb as compared to controls (55.0+/-3.1%; 0.99+/-0.03; 5.0+/-0.2). In conclusion, chronic, moderate hind limb ischemia with consistently reduced perfusion levels persisting over a prolonged period can be established reliably in rnu athymic nude rats and is responsive to pro-angiogenic treatments such as EPC transplantation. This study provides a detailed protocol of a highly reproducible reference model to test novel therapeutic options for limb ischemia.

Local gene transfer and expression following intramuscular administration of FGF-1 plasmid DNA in patients with critical limb ischemia

NV1FGF is an expression plasmid encoding sp.FGF-1(21-154) currently under investigation for therapeutic angiogenesis in clinical trials. NV1FGF plasmid distribution and transgene expression following intramuscular (IM) injection in patients is unknown. The study involved six patients with chronic critical limb ischemia (CLI) planned to undergo amputation. A total dose of 0.5, 2, or 4 mg NV1FGF was administered as eight IM injections (0.006, 0.25, or 0.5 mg per injection) 3-5 days before amputation. Injected sites (30 cm(3)) were divided into equally sized smaller pieces to assess spatial distribution of NV1FGF sequences (PCR), NV1FGF mRNA (reverse transcriptase-PCR), and fibroblast growth factor-1 (FGF-1)-expressing cells (immunohistochemistry). Data indicated gene expression at all doses. The distribution area was within 5-12 cm for NV1FGF sequences containing the expression cassette, up to 5 cm for NV1FGF mRNA, and up to 3 cm for FGF-1-expressing myofibers. All FGF receptors were detected indicating robust potential for bioactivity after NV1FGF gene transfer. Circulating levels of NV1FGF sequences were shown to decrease within days after injection. Data support demonstration of plasmid-mediated gene transfer and expression in muscles from patients with CLI. FGF-1 expression was shown to be limited to injection sites, which supports the concept of multiple-site injection for therapeutic use.

Clinically-driven need for secondary interventions after endovascular revascularization of tibial arteries in patients with critical limb ischemia

PURPOSE To assess the need for clinically-driven secondary revascularization in critical limb ischemia (CLI) patients subsequent to tibial angioplasty during a 2-year follow-up. METHODS Between 2008 and 2010, a total of 128 consecutive CLI patients (80 men; mean age 76.5±9.8 years) underwent tibial angioplasty in 139 limbs. Rutherford categories, ankle-brachial index measurements, and lower limb oscillometries were prospectively assessed. All patients were followed at 3, 6, 12 months, and annually thereafter. Rates of death, primary and secondary sustained clinical improvement, target lesion (TLR) and target extremity revascularization (TER), as well as major amputation, were analyzed retrospectively. Primary clinical improvement was defined as improvement in Rutherford category to a level of intermittent claudication without unplanned amputation or TLR. RESULTS All-cause mortality was 8.6%, 14.8%, 22.9%, and 29.1% at 3, 6, 12, and 24 months. At the same intervals, rates of primary sustained clinical improvement were 74.5%, 53.0%, 42.7%, and 37.1%; for secondary improvement, the rates were 89.1%, 76.0%, 68.4%, and 65.0%. Clinically-driven TLR rates were 14.6%, 29.1%, 41.6%, 46.2%; the rates for TER were 3.0%, 13.6%, 17.2%, and 27.6% in corresponding intervals, while the rates of major amputation were 1.5%, 5.5%, 10.1%, and 10.1%. CONCLUSION Clinically-driven TLR is frequently required to maintain favorable functional clinical outcomes in CLI patients following tibial angioplasty. Dedicated technologies addressing tibial arterial restenosis warrant further academic scrutiny.

A novel paradigm for patient-cooperative control of upper-limb rehabilitation robots

Early intervention and intensive therapy improve the outcome of neuromuscular rehabilitation. There are indications that where a patient is motivated and premeditates their movement, the recovery is more effective. Therefore, a strategy for patient-cooperative control of rehabilitation devices for upper extremities is proposed and evaluated. The strategy is based on the minimal intervention principle allowing an efficient exploitation of task space redundancies and resulting in user-driven movement trajectories. The patient's effort is taken into consideration by enabling the machine to comply with forces exerted by the user. The interaction is enhanced through a multimodal display and a virtually generated environment that includes haptic, visual and sound modalities.

ΔNp63 REGULATES A COMPLEX NETWORK OF TARGET GENES IN LIMB AND EPIDERMAL DEVELOPMENT

The skin is composed of two major compartments, the dermis and epidermis. The epidermis forms a barrier to protect the body. The stratified epithelium has self-renewing capacity throughout life, and continuous turnover is mediated by stem cells in the basal layer. p63 is structurally and functionally related to p53. In spite of their structural similarities, p63 is critical for the development and maintenance of stratified epithelial tissues, unlike p53. p63 is highly expressed in the epidermis and previously has been shown to play a critical role in the development and maintenance of the epidermis. The study of p63 has been complicated due to the existence of multiple isoforms: those with a transactivation domain (TAp63) and those lacking this domain (ΔNp63). Mice lacking p63 cannot form skin, have craniofacial and skeletal defects and die within hours after birth. These defects are due to the ability of p63 to regulate multiple processes in skin development including epithelial stem cell proliferation, differentiation, and adherence programs. To determine the roles of these isoforms in skin development and maintenance, isoform specific p63 conditional knock out mice were generated by our lab. TAp63-/- mice age prematurely, develop blisters, and display wound-healing defects that result from hyperproliferation of dermal stem cells. That results in premature depletion of these cells, which are necessary for wound repair, that indicates TAp63 plays a role in dermal/epidermal maintenance. To study the role of ΔNp63, I generated a ΔNp63-/- mouse and analyzed the skin by performing immunofluorescence for markers of epithelial differentiation. The ΔNp63-/- mice developed a thin, disorganized epithelium but differentiation markers were expressed. Interestingly, the epidermis from ΔNp63-/- mice co-expressed K14 and K10 in the same cell suggesting defects in epidermal differentiation and stratification. This phenotype is reminiscent of the DGCR8fl/fl;K14Cre and Dicerfl/fl;K14Cre mice skin. Importantly, DGCR8-/- embryonic stem cells (ESCs) display a hyperproliferation defect by failure to silence pluripotency genes. Furthermore, I have observed that epidermal cells lacking ΔNp63 display a phenotype reminiscent of embryonic stem cells instead of keratinocytes. Thus, I hypothesize that genes involved in maintaining pluripotency, like Oct4, may be upregulated in the absence of ΔNp63. To test this, q-RT PCR was performed for Oct4 mRNA with wild type and ΔNp63-/- 18.5dpc embryo skin. I found that the level of Oct4 was dramatically increased in the absence of ΔNp63-/-. Based on these results, I hypothesized that ΔNp63 induces differentiation by silencing pluripotency regulators, Oct4, Sox2 and Nanog directly through the regulation of DGCR8. I found that DGCR8 restoration resulted in repression of Oct4, Sox2 and Nanog in ΔNp63-/- epidermal cells and rescue differentiation defects. Loss of ΔNp63 resulted in pluripotency that caused defect in proper differentiation and stem cell like phenotype. This led me to culture the ΔNp63-/- epidermal cells in neuronal cell culture media in order to address whether restoration of DGCR8 can transform epidermal cells to neuronal cells. I found that DGCR8 restoration resulted in a change in cell fate. I also found that miR470 and miR145 play a role in the induction of pluripotency by repressing Oct4, Sox2 and Nanog. This indicates that ΔNp63 induces terminal differentiation through the regulation of DGCR8.

Early recoil after balloon angioplasty of tibial artery obstructions in patients with critical limb ischemia.

PURPOSE To assess the extent of early recoil in patients with critical limb ischemia (CLI) undergoing conventional tibial balloon angioplasty. METHODS Our hypothesis was that early recoil, defined as lumen compromise >10%, is frequent and accounts for considerable luminal narrowing after tibial angioplasty, promoting restenosis. To test this theory, 30 consecutive CLI patients (18 men; mean age 76.2±12.1 years) were angiographically evaluated immediately after tibial balloon angioplasty and 15 minutes later. Half the patients were diabetics. Target lesions included anterior and posterior tibial arteries and the peroneal artery with / without the tibioperoneal trunk. Mean tibial lesion length was 83.8 mm. Early elastic recoil was determined on the basis of minimal lumen diameter (MLD) measurements at baseline (MLDbaseline), immediately after tibial balloon angioplasty (MLDpostdilation), and 15 minutes thereafter (MLD15min). RESULTS Elastic recoil was observed in 29 (97%) patients with a mean luminal compromise of 29% according to MLD measurements (MLDbaseline 0.23 mm, MLD postdilation 2.0 mm, and MLD15min 1.47 mm). CONCLUSION Early recoil is frequently observed in CLI patients undergoing tibial angioplasty and may significantly contribute to restenosis. These findings support the role of dedicated mechanical scaffolding approaches for the prevention of restenosis in tibial arteries.

IN.PACT Amphirion paclitaxel eluting balloon versus standard percutaneous transluminal angioplasty for infrapopliteal revascularization of critical limb ischemia: rationale and protocol for an ongoing randomized controlled trial

BACKGROUND The effectiveness and durability of endovascular revascularization therapies for chronic critical limb ischemia (CLI) are challenged by the extensive burden of infrapopliteal arterial disease and lesion-related characteristics (e.g., severe calcification, chronic total occlusions), which frequently result in poor clinical outcomes. While infrapopliteal vessel patency directly affects pain relief and wound healing, sustained patency and extravascular care both contribute to the ultimate "patient-centric" outcomes of functional limb preservation, mobility and quality of life (QoL). METHODS/DESIGN IN.PACT DEEP is a 2:1 randomized controlled trial designed to assess the efficacy and safety of infrapopliteal arterial revascularization between the IN.PACT Amphirion™ paclitaxel drug-eluting balloon (IA-DEB) and standard balloon angioplasty (PTA) in patients with Rutherford Class 4-5-6 CLI. DISCUSSION This multicenter trial has enrolled 358 patients at 13 European centers with independent angiographic core lab adjudication of the primary efficacy endpoint of target lesion late luminal loss (LLL) and clinically driven target lesion revascularization (TLR) in major amputation-free surviving patients through 12-months. An independent wound core lab will evaluate all ischemic wounds to assess the extent of healing and time to healing at 1, 6, and 12 months. A QoL questionnaire including a pain scale will assess changes from baseline scores through 12 months. A Clinical Events Committee and Data Safety Monitoring Board will adjudicate the composite primary safety endpoints of all-cause death, major amputation, and clinically driven TLR at 6 months and other trial endpoints and supervise patient safety throughout the study. All patients will be followed for 5 years. A literature review is presented of the current status of endovascular treatment of CLI with drug-eluting balloon and standard PTA. The rationale and design of the IN.PACT DEEP Trial are discussed. IN.PACT DEEP is a milestone, prospective, randomized, robust, independent core lab-adjudicated CLI trial that will evaluate the role of a new infrapopliteal revascularization technology, the IA-DEB, compared to PTA. It will assess the overall impact on infrapopliteal artery patency, limb salvage, wound healing, pain control, QoL, and patient mobility. The 1-year results of the adjudicated co-primary and secondary endpoints will be available in 2014. TRIAL REGISTRATION NCT00941733

Sleep-Disordered Breathing and Periodic Limb Movements in Narcolepsy with Cataplexy: A Systematic Analysis of 35 Consecutive Patients

Background: Disturbed sleep is a core feature of narcolepsy with cataplexy (NC). Few studies have independently assessed sleep-disordered breathing (SDB) and periodic limb movements (PLMs) in non-homogeneous series of patients with and without cataplexy. We systematically assessed both SDB and PLMs in well-defined NC patients. Methods: We analyzed the clinical and polysomnographic features of 35 consecutive NC patients (mean age 40 ± 16 years, 51% males, 23/23 hypocretin-deficient) to assess the prevalence of SDB (apnea-hypopnea index >5) and PLMs (periodic leg movements in sleep (PLMI) >15) together with their impact on nocturnal sleep and daytime sleepiness using the multiple sleep latency test. Results: 11 (31%) and 14 (40%) patients had SDB and PLMs, respectively. SDB was associated with older age (49 ± 16 vs. 35 ± 13 years, p = 0.02), higher BMI (30 ± 5 vs. 27 ± 6, p = 0.05), and a trend towards higher PLMI (25 ± 20 vs. 12 ± 23, p = 0.052), whereas PLMs with older age (50 ± 16 vs. 33 ± 11 years, p = 0.002) and reduced and fragmented sleep (e.g. sleep efficiency of 82 ± 12% vs. 91 ± 6%, p = 0.015; sleep time of 353 ± 66 vs. 395 ± 28, p = 0.010). SDB and PLMs were also mutually associated (p = 0.007), but not correlated to daytime sleepiness. Conclusions: SDB and PLMs are highly prevalent and associated in NC. Nevertheless, SDB and PLMs are rarely severe, suggesting an overall limited effect on clinical manifestations.