Multispectral Optoacoustic Tomography of Matrix Metalloproteinase Activity in Vulnerable Human Carotid Plaques

Elevated expression of cathepsins, integrins and matrix metalloproteinases (MMPs) is typically associated with atherosclerotic plaque instability. While fluorescent tagging of such molecules has been amply demonstrated, no imaging method was so far shown capable of resolving these inflammation-associated tags with high fidelity and resolution beyond microscopic depths. This study is aimed at demonstrating a new method with high potential for noninvasive clinical cardiovascular diagnostics of vulnerable plaques using high-resolution deep-tissue multispectral optoacoustic tomography (MSOT) technology. MMP-sensitive activatable fluorescent probe (MMPSense (TM) 680) was applied to human carotid plaques from symptomatic patients. Atherosclerotic activity was detected by tuning MSOT wavelengths to activation-dependent absorption changes of the molecules, structurally modified in the presence of enzymes. MSOT analysis simultaneously provided morphology along with heterogeneous MMP activity with better than 200 micron resolution throughout the intact plaque tissue. The results corresponded well with epi-fluorescence images made from thin cryosections. Elevated MMP activity was further confirmed by zymography, accompanied by increased macrophage influx. We demonstrated, for the first time to our knowledge, the ability of MSOT to provide volumetric images of activatable molecular probe distribution deep within optically diffuse tissues. High-resolution mapping of MMP activity was achieved deep in the vulnerable plaque of intact human carotid specimens. This performance directly relates to pre-clinical screening applications in animal models and to clinical decision potential as it might eventually allow for highly specific visualization and staging of plaque vulnerability thus impacting therapeutic clinical decision making

Distribution of Matrix Metalloproteinases in Human Atherosclerotic Carotid Plaques and Their Production by Smooth Muscle Cells and Macrophage Subsets

In this study, the potential of matrix metalloproteinase (MMP) sense for detection of atherosclerotic plaque instability was explored. Secondly, expression of MMPs by macrophage subtypes and smooth muscle cells (SMCs) was investigated. Twenty-three consecutive plaques removed during carotid endarterectomy were incubated in MMPSense (TM) 680 and imaged with IVISA (R) Spectrum. mRNA levels of MMPs, macrophage markers, and SMCs were determined in plaque specimens, and in in vitro differentiated M1 and M2 macrophages. There was a significant difference between autofluorescence signals and MMPSense signals, both on the intraluminal and extraluminal sides of plaques. MMP-9 and CD68 messenger RNA (mRNA) expression was higher in hot spots, whereas MMP-2 and alpha SMA expression was higher in cold spots. In vitro M2 macrophages had higher mRNA expression of MMP-1, MMP-9, MMP-12, and TIMP-1 compared to M1 macrophages. MMP-9 is most dominantly MMP present in atherosclerotic plaques and is produced by M2 rather than M1 macrophages

Intraoperative Multispectral Fluorescence Imaging for the Detection of the Sentinel Lymph Node in Cervical Cancer: A Novel Concept

PURPOSE: Real-time intraoperative near-infrared fluorescence (NIRF) imaging is a promising technique for lymphatic mapping and sentinel lymph node (SLN) detection. The purpose of this technical feasibility pilot study was to evaluate the applicability of NIRF imaging with indocyanin green (ICG) for the detection of the SLN in cervical cancer. PROCEDURES: In ten patients with early stage cervical cancer, a mixture of patent blue and ICG was injected into the cervix uteri during surgery. Real-time color and fluorescence videos and images were acquired using a custom-made multispectral fluorescence camera system. RESULTS: Real-time fluorescence lymphatic mapping was observed in vivo in six patients; a total of nine SLNs were detected, of which one (11%) contained metastases. Ex vivo fluorescence imaging revealed the remaining fluorescent signal in 11 of 197 non-sentinel LNs (5%), of which one contained metastatic tumor tissue. None of the non-fluorescent LNs contained metastases. CONCLUSIONS: We conclude that lymphatic mapping and detection of the SLN in cervical cancer using intraoperative NIRF imaging is technically feasible. However, the technique needs to be refined for full applicability in cervical cancer in terms of sensitivity and specificity

Description of orthotic properties and effect evaluation of ankle-foot orthoses in non-spastic calf muscle weakness

Objective: To describe the orthotic properties and evaluate the effects of ankle-foot orthoses for calf muscle weakness in persons with non-spastic neuromuscular disorders compared with shoes-only. Design: Cross-sectional study. Subjects: Thirty-four persons who used ankle-foot orthoses for non-spastic calf muscle weakness. Methods: The following orthotic properties were measured: ankle-foot orthosis type, mass, and ankle and footplate stiffness. For walking with shoes- only and with the ankle-foot orthoses, walking speed, energy cost and gait biomechanics were assessed. Results: Four types of ankle-foot orthosis were identified: shaft-reinforced orthopaedic shoes (n = 6), ventral ankle-foot orthoses (n = 10), dorsal leaf ankle-foot orthoses (n = 12) and dorsiflexion-stop ankle-foot orthoses (n = 6). These types differed significantly with regards to mass, ankle-and footplate stiffness. Compared with shoes-only, all anklefoot orthoses/orthopaedic shoes groups combined increased walking speed by 0.18 m/s (95% confidence interval (95% CI) 0.13-0.23), reduced energy cost by 0.70 J/kg/m (95% CI 0.48-0.94) and limited ankle dorsiflexion by -3.0° (95% CI 1.3-4.7). Higher ankle-foot orthoses ankle stiffness correlated with greater reductions in walking energy cost and maximal ankle dorsiflexion angle. Conclusion: Ankle-foot orthoses for persons with non-spastic calf muscle weakness vary greatly in properties and effects on gait. The large variation in effectiveness may be due to differences in ankle stiffness, although this requires further prospective evaluation.</p

Precision orthotics: optimising ankle foot orthoses to improve gait in patients with neuromuscular diseases; protocol of the PROOF-AFO study, a prospective intervention study

In patients with neuromuscular disorders and subsequent calf muscle weakness, metabolic walking energy cost (EC) is nearly always increased, which may restrict walking activity in daily life. To reduce walking EC, a spring-like ankle-foot-orthosis (AFO) can be prescribed. However, the reduction in EC that can be obtained from these AFOs is stiffness dependent, and it is unknown which AFO stiffness would optimally support calf muscle weakness. The PROOF-AFO study aims to determine the effectiveness of stiffness-optimised AFOs on reducing walking EC, and improving gait biomechanics and walking speed in patients with calf muscle weakness, compared to standard, non-optimised AFOs. A second aim is to build a model to predict optimal AFO stiffness. A prospective intervention study will be conducted. In total, 37 patients with calf muscle weakness who already use an AFO will be recruited. At study entry, participants will receive a new custom-made spring-like AFO of which the stiffness can be varied. For each patient, walking EC (primary outcome), gait biomechanics and walking speed (secondary outcomes) will be assessed for five stiffness configurations and the patient's own (standard) AFO. On the basis of walking EC and gait biomechanics outcomes, the optimal AFO stiffness will be determined. After wearing this optimal AFO for 3 months, walking EC, gait biomechanics and walking speed will be assessed again and compared to the standard AFO. The Medical Ethics Committee of the Academic Medical Centre in Amsterdam has approved the study protocol. The study is registered at the Dutch trial register (NTR 5170). The PROOF-AFO study is the first to compare stiffness-optimised AFOs with usual care AFOs in patients with calf muscle weakness. The results will also provide insight into factors that influence optimal AFO stiffness in these patients. The results are necessary for improving orthotic treatment and will be disseminated through international peer-reviewed journals and scientific conference

Description of orthotic properties and effect evaluation of ankle-foot orthoses in non-spastic calf muscle weakness

OBJECTIVE: To describe the orthotic properties and evaluate the effects of ankle-foot orthoses for calf muscle weakness in persons with non-spastic neuromuscular disorders compared with shoes-only. DESIGN: Cross-sectional study. SUBJECTS: Thirty-four persons who used ankle-foot orthoses for non-spastic calf muscle weakness. METHODS: The following orthotic properties were measured: ankle-foot orthosis type, mass, and ankle and footplate stiffness. For walking with shoes-only and with the ankle-foot orthoses, walking speed, energy cost and gait biomechanics were assessed. RESULTS: Four types of ankle-foot orthosis were identified: shaft-reinforced orthopaedic shoes (n = 6), ventral ankle-foot orthoses (n = 10), dorsal leaf ankle-foot orthoses (n = 12) and dorsiflexion-stop ankle-foot orthoses (n = 6). These types differed significantly with regards to mass, ankle-and footplate stiffness. Compared with shoes-only, all ankle-foot orthoses/orthopaedic shoes groups combined increased walking speed by 0.18 m/s (95% confidence interval (95% CI) 0.13-0.23), reduced energy cost by 0.70 J/kg/m (95% CI 0.48-0.94) and limited ankle dorsiflexion by -3.0° (95% CI 1.3-4.7). Higher ankle-foot orthoses ankle stiffness correlated with greater reductions in walking energy cost and maximal ankle dorsiflexion angle. CONCLUSION: Ankle-foot orthoses for persons with non-spastic calf muscle weakness vary greatly in properties and effects on gait. The large variation in effectiveness may be due to differences in ankle stiffness, although this requires further prospective evaluation

Compensations in lower limb joint work during walking in response to unilateral calf muscle weakness

Background: Patients with calf muscle weakness due to neuromuscular disorders have a reduced ankle push-off work, which leads to increased energy dissipation at contralateral heel-strike. Consequently, compensatory positive work needs to be generated, which is mechanically less efficient. It is unknown whether neuromuscular disorder patients compensate with their ipsilateral hip and/or contralateral leg; and if such compensatory joint work is related to walking energy cost. Research question: Do patients with calf muscle weakness compensate for the increase in negative joint work by increasing positive ipsilateral hip work and/or positive contralateral leg work? And is the total mechanical work related with walking energy cost? Methods: Seventeen patients with unilateral flaccid calf muscle weakness and 10 healthy individuals performed the following two tests: i) a barefoot 3D gait analysis at comfortable speed and matched control speed (i.e. 0.4 non-dimensional) to assess lower limb joint work and ii) a 6-minute walk test at comfortable speed to assess walking energy cost. Results: Patients had a lower comfortable walking speed compared to healthy individuals (1.05 vs 1.36 m/s, p &lt; 0.001) and did not increase positive lower limb joint work at comfortable speed. At matched speed (1.25 m/s), patients showed increased positive work at their ipsilateral hip (0.38 ± 0.08 vs 0.27 ± 0.07, p = 0.001) and/or contralateral leg (0.99 ± 0.14 vs 0.69 ± 0.14, p &lt; 0.001). Patients with weakest plantar flexors used both strategies. No relation between total positive work and walking energy cost was found (r = 0.43, p = 0.122). Significance: Patients with unilateral calf muscle weakness compensated for reduced ankle push-off work by lowering their comfortable walking speed or, at matched speed, by generating additional positive joint work at the ipsilateral hip and/or contralateral leg. The additional positive joint work at matched speed did not explain the elevated walking energy cost at comfortable speed, which needs further exploration.</p

Description of orthotic properties and effect evaluation of ankle-foot orthoses in non-spastic calf muscle weakness

Objective: To describe the orthotic properties and evaluate the effects of ankle-foot orthoses for calf muscle weakness in persons with non-spastic neuromuscular disorders compared with shoes-only. Design: Cross-sectional study. Subjects: Thirty-four persons who used ankle-foot orthoses for non-spastic calf muscle weakness. Methods: The following orthotic properties were measured: ankle-foot orthosis type, mass, and ankle and footplate stiffness. For walking with shoes- only and with the ankle-foot orthoses, walking speed, energy cost and gait biomechanics were assessed. Results: Four types of ankle-foot orthosis were identified: shaft-reinforced orthopaedic shoes (n = 6), ventral ankle-foot orthoses (n = 10), dorsal leaf ankle-foot orthoses (n = 12) and dorsiflexion-stop ankle-foot orthoses (n = 6). These types differed significantly with regards to mass, ankle-and footplate stiffness. Compared with shoes-only, all anklefoot orthoses/orthopaedic shoes groups combined increased walking speed by 0.18 m/s (95% confidence interval (95% CI) 0.13-0.23), reduced energy cost by 0.70 J/kg/m (95% CI 0.48-0.94) and limited ankle dorsiflexion by -3.0° (95% CI 1.3-4.7). Higher ankle-foot orthoses ankle stiffness correlated with greater reductions in walking energy cost and maximal ankle dorsiflexion angle. Conclusion: Ankle-foot orthoses for persons with non-spastic calf muscle weakness vary greatly in properties and effects on gait. The large variation in effectiveness may be due to differences in ankle stiffness, although this requires further prospective evaluation.Biomechatronics & Human-Machine Contro

Modifying ankle foot orthosis stiffness in patients with calf muscle weakness: gait responses on group and individual level

BACKGROUND: To improve gait, persons with calf muscle weakness can be provided with a dorsal leaf spring ankle foot orthosis (DLS-AFO). These AFOs can store energy during stance and return this energy during push-off, which, in turn, reduces walking energy cost. Simulations indicate that the effect of the DLS-AFO on walking energy cost and gait biomechanics depends on its stiffness and on patient characteristics. We therefore studied the effect of varying DLS-AFO stiffness on reducing walking energy cost, and improving gait biomechanics and AFO generated power in persons with non-spastic calf muscle weakness, and whether the optimal AFO stiffness for maximally reducing walking energy cost varies between persons. METHODS: Thirty-seven individuals with neuromuscular disorders and non-spastic calf muscle weakness were included. Participants were provided with a DLS-AFO of which the stiffness could be varied. For 5 stiffness configurations (ranging from 2.8 to 6.6 Nm/degree), walking energy cost (J/kg/m) was assessed using a 6-min comfortable walk test. Selected gait parameters, e.g. maximal dorsiflexion angle, ankle power, knee angle, knee moment and AFO generated power, were derived from 3D gait analysis. RESULTS: On group level, no significant effect of DLS-AFO stiffness on reducing walking energy cost was found (p = 0.059, largest difference: 0.14 J/kg/m). The AFO stiffness that reduced energy cost the most varied between persons. The difference in energy cost between the least and most efficient AFO stiffness was on average 10.7%. Regarding gait biomechanics, increasing AFO stiffness significantly decreased maximal ankle dorsiflexion angle (- 1.1 ± 0.1 degrees per 1 Nm/degree, p &lt; 0.001) and peak ankle power (- 0.09 ± 0.01 W/kg, p &lt; 0.001). The reduction in minimal knee angle (- 0.3 ± 0.1 degrees, p = 0.034), and increment in external knee extension moment in stance (- 0.01 ± 0.01 Nm/kg, p = 0.016) were small, although all stiffness' substantially affected knee angle and knee moment compared to shoes only. No effect of stiffness on AFO generated power was found (p = 0.900). CONCLUSIONS: The optimal efficient DLS-AFO stiffness varied largely between persons with non-spastic calf muscle weakness. Results indicate this is caused by an individual trade-off between ankle angle and ankle power affected differently by AFO stiffness. We therefore recommend that the AFO stiffness should be individually optimized to best improve gait. TRIAL REGISTRATION NUMBER: Nederlands Trial Register 5170. Registration date: May 7th 2015. http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=5170.Biomechatronics & Human-Machine Contro