In vivo multimodal imaging of hyaluronan-mediated inflammatory response in articular cartilage.

OBJECTIVE: One driving factor in the progression to posttraumatic osteoarthritis (PTOA) is the perpetuation of the inflammatory response to injury into chronic inflammation. Molecular imaging offers many opportunities to complement the sensitivity of current imaging modalities with molecular specificity. The goal of this study was to develop and characterize agents to image hyaluronan (HA)-mediated inflammatory signaling. DESIGN: We developed optical (Cy5.5-P15-1) and magnetic resonance contrast agents (Gd-DOTA-P15-1) based in a hyaluronan-binding peptide (P15-1) that has shown anti-inflammatory effects on human chondrocytes, and validated them in vitro and in vivo in two animal models of PTOA. RESULTS: In vitro studies with a near infrared (NIR) Cy5.5-P15-1 imaging agent showed a fast and stable localization of Cy5.5-P15-1 on chondrocytes, but not in synovial cells. In vivo NIR showed significantly higher retention of imaging agent in PTOA knees between 12 and 72 h (n = 8, Cohen's d > 2 after 24 h). NIR fluorescence accumulation correlated with histologic severity in cartilage and meniscus (ρ between 0.37 and 0.57, P < 0.001). By using in vivo magnetic resonance imaging with a Gd-DOTA-P15-1 contrast agent in 12 rats, we detected a significant decrease of T1 on injured knees in all cartilage plates at 48 h (-15%, 95%-confidence interval (CI) = [-18%,-11%]) while no change was observed in the controls (-2%, 95%-CI = [-5%,+1%]). CONCLUSIONS: This study provides the first in vivo evidence that hyaluronan-related inflammatory response in cartilage after injury is a common finding. Beyond P15-1, we have demonstrated that molecular imaging can provide a versatile technology to investigate and phenotype PTOA pathogenesis, as well as study therapeutic interventions.

A novel rat model for subchondral microdamage in acute knee injury: a potential mechanism in post-traumatic osteoarthritis.

OBJECTIVE: Subchondral microdamage may play an important role in post-traumatic osteoarthritis (PTOA) development following anterior cruciate ligament (ACL) rupture. It remains unknown whether this injury mechanism causes subchondral microdamage, or whether its repair occurs by targeted osteoclast-mediated remodeling. If so these events may represent a mechanism by which subchondral bone is involved in PTOA. Our objective was to test the hypothesis that subchondral microdamage occurs, and is co-localized with remodeling, in a novel rat model of ACL rupture. DESIGN: We developed a novel non-invasive rat animal model for ACL rupture and subchondral microdamage generation. By inducing ACL rupture noninvasively rather than surgically, this more closely mimics the clinical injury. MicroCT, MRI and histological methods were used to measure microstructural changes, ligament damage, and cellular/matrix degeneration, respectively. RESULTS: We reproducibly generated ACL rupture without damage to other soft joint tissues. Immediately after injury, increased microdamage was found in the postero-medial aspect of the tibia. Microstructural parameters showed increased resorption at 2 weeks, which returned to baseline. Dynamic histomorphometry showed increased calcein label uptake in the same region at 4 and 8 weeks. Chondrocyte death and protease activity in cartilage was also noted, however whether this was directly linked to subchondral changes is not yet known. Similarly, cartilage scoring showed degradation at 4 and 8 weeks post-injury. CONCLUSIONS: This study shows that our novel model can be used to study subchondral microdamage after ACL-rupture, and its association with localized remodeling. Cartilage degeneration, on a similar time-scale to other models, is also a feature of this system.

Topographic deformation patterns of knee cartilage after exercises with high knee flexion: an in vivo 3D MRI study using voxel-based analysis at 3T.

OBJECTIVES: To implement a novel voxel-based technique to identify statistically significant local cartilage deformation and analyze in-vivo topographic knee cartilage deformation patterns using a voxel-based thickness map approach for high-flexion postures. METHODS: Sagittal 3T 3D-T1w-FLASH-WE-sequences of 10 healthy knees were acquired before and immediately after loading (kneeling/squatting/heel sitting/knee bends). After cartilage segmentation, 3D-reconstruction and 3D-registration, colour-coded deformation maps were generated by voxel-based subtraction of loaded from unloaded datasets to visualize cartilage thickness changes in all knee compartments. RESULTS: Compression areas were found bifocal at the peripheral medial/caudolateral patella, both posterior femoral condyles and both anterior/central tibiae. Local cartilage thickening were found adjacent to the compression areas. Significant local strain ranged from +13 to -15 %. Changes were most pronounced after squatting, least after knee bends. Shape and location of deformation areas varied slightly with the loading paradigm, but followed a similar pattern consistent between different individuals. CONCLUSIONS: Voxel-based deformation maps identify individual in-vivo load-specific and posture-associated strain distribution in the articular cartilage. The data facilitate understanding individual knee loading properties and contribute to improve biomechanical 3 models. They lay a base to investigate the relationship between cartilage degeneration patterns in common osteoarthritis and areas at risk of cartilage wear due to mechanical loading in work-related activities. KEY POINTS: • 3D MRI helps differentiate true knee-cartilage deformation from random measurement error • 3D MRI maps depict in vivo topographic distribution of cartilage deformation after loading • 3D MRI maps depict in vivo intensity of cartilage deformation after loading • Locating cartilage contact areas might aid differentiating common and work-related osteoarthritis.

Feasibility of a RARE-based sequence for quantitative diffusion-weighted MRI of the spine.

The feasibility of a diffusion-weighted single-shot fast-spin-echo sequence for the diagnostic work-up of bone marrow diseases was assessed. Twenty healthy controls and 16 patients with various bone marrow pathologies of the spine (bone marrow edema, tumor and inflammation) were examined with a diffusion-weighted single-shot sequence based on a modified rapid acquisition with relaxation enhancement (mRARE) technique; four diffusion weightings (b-values: 50, 250, 500 and 750 s/mm(2)) in three orthogonal orientations were applied. Apparent diffusion coefficients (ADCs) were determined in the bone marrow and in the intervertebral discs of healthy volunteers and in diseased bone marrow. Ten of the 20 volunteers were repeatedly scanned within 30 min to examine short-time reproducibility. Spatial reproducibility was assessed by measuring ADCs in two different slices including the same lesion in 12 patients. The ADCs of the lesions exhibited significantly higher values, (1.27 +/- 0.32)x10(-3) mm(2)/s, compared with healthy bone marrow, (0.21 +/- 0.10)x10(-3) mm(2)/s. Short-time and spatial reproducibility had a mean coefficient of variation of 2.1% and 6.4%, respectively. The diffusion-weighted mRARE sequence provides a reliable tool for determining quantitative ADCs in vertebral bone marrow with adequate image quality.

[T2 relaxation time in patellar cartilage--global and regional reproducibility at 1.5 tesla and 3 tesla]. / T2-Relaxationszeit am Patellaknorpel--Globale und regionale Reproduzierbarkeit bei 1,5 Tesla und 3 Tesla.

PURPOSE: Evaluation of the global and regional reproducibility of T2 relaxation time in patellar cartilage at 1.5 T and 3 T. MATERIALS AND METHODS: 6 left patellae of 6 healthy volunteers (aged 25 - 30, 3 female, 3 male) were examined using a fat-saturated multiecho sequence and a T1-w 3D-FLASH sequence with water excitation at 1.5 Tesla and 3 Tesla. Three consecutive data sets were acquired within one MRI session with the examined knee being repositioned in the coil and scanner between each data set. The segmented cartilage (FLASH sequence) was overlaid on the multiecho data and T2 values were calculated for the total cartilage, 3 horizontal layers consisting of a superficial, intermedial and deep layer, 3 facets consisting of a medial, median (ridge) and lateral facet (global T2 values) and 27 ROIs/MRI slices (regional T2 value). The reproducibility (precision error) was calculated as the root mean square average of the individual standard deviations [ms] and coefficients of variation (COV) [%]. RESULTS: The mean global reproducibility error for T2 was 3.53 % (+/- 0.38 %) at 1.5 Tesla and 3.25 % (+/- 0.61 %) at 3 Tesla. The mean regional reproducibility error for T2 was 8.62 % (+/- 2.61 %) at 1.5 Tesla and 9.66 % (+/- 3.37 %) at 3 Tesla. There was no significant difference with respect to absolute reproducibility errors between 1.5 Tesla and 3 Tesla at a constant spatial resolution. However, different reproducibility errors were found between the cartilage layers. One third of the data variability could be attributed to the influence of the different cartilage layers, and another 10 % to the influence of the separate MRI slices. CONCLUSION: Our data provides an estimation of the global and regional reproducibility errors of T2 in healthy cartilage. In the analysis of small subregions, an increase in the regional reproducibility error must be accepted. The data may serve as a basis for sample size calculations of study populations and may contribute to the decision regarding the level of detail of an evaluation of study data.

Techniques for diffusion-weighted imaging of bone marrow.

Diffusion-weighted magnetic resonance imaging (DWI) is an imaging technique which is sensitive to random water movements in spatial scales far below those typically accessible by magnetic resonance imaging (MRI). This property makes DWI a powerful tool for diagnosis of diseases which involve alterations in water mobility, such as acute stroke. In bone marrow, DWI has been proven to be a highly useful method for the differential diagnosis of benign and malignant compression fractures. Unfortunately, the application of DWI sequences to the bone marrow frequently suffers from artifacts, which in some cases seriously restrict the diagnostic utility of the image. This requires the introduction of additional correction techniques, or even the development of new sequences. Thus, the selection of an adequate imaging technique for DWI of the bone marrow is a very important issue. In this article the most important sequences for DWI of the bone marrow are reviewed. Special attention is paid to the problems associated with these sequences, as well as their possible solutions.

Sustained firing of alpha and gamma hind limb motoneurons induced by stimulation of the pudendal nerve.

Axons from receptors in the cat vaginal wall run in the sensory pudendal nerve (SPN), and brief (<10 s) vaginal probing (VP) in the decerebrate cat produces a long-lasting (>1 min) contraction of the triceps surae (TS) muscles. The aim of the present project was to find out whether brief SPN stimulation also produces sustained TS response and, eventually, to study the mechanisms involved in it. Decerebrate female cats were used. In some cats, TS electromyography (EMG) and tension response were recorded; stimulation of left SPN with single or repetitive trains of shocks produced a bilateral TS response that outlasted the stimulus >1 min as VP did. In paralyzed cats (pancuronium; Panc), intracellular recordings were made from hind limb motoneurons (MNs). SPN stimulation produced a depolarization 1 min) electroneurographic (ENG) postdischarge in a small filament of the medial gastrocnemius (MG) nerve; the MG EMG postdischarge was also recorded. Large spikes (LS) and small spikes (SS) were distinguished in the ENG. During the postdischarge, LS frequency and the integrated EMG activity correlated well (r > 0.9); no correlation was found between SS and EMG. After Panc injection, LS postdischarge was absent but the SS postdischarge remained. LS followed by EMG potential were also evoked by brief TS stretch (reflex LS); single shocks to SPN only elicited SS that were not followed by EMG potential. It is concluded that alpha axons and gamma axons produced LS and SS, respectively, and that SPN activates gamma axons. It is proposed that, in the nonparalyzed cats, the stimulation of SPN with trains of shocks might cause an increase in the afferent inflow from muscle spindles to alpha MNs through the sustained firing of gamma MNs. The increased excitatory inflow would depolarize alpha MNs and allow bistable MN firing; Panc would decrease this inflow by blocking transmission to the spindle fibers.

Methods to find aponeurosis and tendon stiffness and the onset of muscle contraction.

A method to measure small movements of living tissues either large or small is presented. The method is based on the detection of changes in reflected infrared light. An optocoupler (coupled photodiode and photodetector) and a small (< 1 cm2) mirror were used. The optocoupler (OC) has a low cost and it can be calibrated easily. It can be also used as the transducer of a strain-gage. Three different uses are shown: (a) as a strain-gage transducer; (b) detection of tendon and aponeurosis movements in large muscles (cat soleus); (c) detection of the onset of muscle contraction. Movements of less than 1 microm can be detected with the aid of automatic averaging of the signals. Concerning the second use (b), it permits the estimation of tendon stretch. Concerning the third use, the onset of muscle movement precedes by at least 2 ms that of the force recorded at the tendon.

The cat pudendal nerve: afferent fibers responding to mechanical stimulation of the perineal skin, the vagina or the uterine cervix.

Some afferent fibers from the pudendal nerve of the female cat were stimulated by pressing on the perineal skin, the vagina or the uterine cervix. Three different types of skin mechanoreceptors were found: (1) with low threshold (< 20 mg) and slow-adapting discharges; (2) with high threshold (0.1-0.5 g) and slow-adapting discharges; and (3) with low threshold and fast-adapting discharges. Most of these receptors increased their firing frequency as the velocity of skin indentation was increased (velocity detectors). The average conduction velocity of the skin afferents was 29 +/- 9 m/s. The receptors located at the vagina showed a fast-adapting response to probing and were sensitive to the velocity of the probe movement. Most of these receptors, however, showed a slow adaptation when the vaginal wall was distended with a balloon. The conduction velocity in vaginal afferents was 37 +/- 16 m/s. Those receptors responding to pressure on the uterine cervix adapted slowly to constant pressure but were sensitive to the velocity of the pressure pulses. The conduction velocity in the afferents from the uterine cervix was 31 +/- 9 m/s.

Sustained activation of the triceps surae muscles produced by mechanical stimulation of the genital tract of the female cat.

In decerebrate cats, controlled mechanical stimulation of the perivulvar skin, the vaginal wall or the cervix uteri induced visible hind limb extension. Pressing on the cervix uteri produced the greater response. To quantify these responses, the EMG activity and the tension developed by the normally inserted triceps surae muscles were recorded. The activity induced in these muscles by stimulation of the genital canal outlasted the stimulus by many seconds or a few minutes. These effects disappeared after spinalization at the T12 level. We propose that stimulation of the vaginal canal in the female cat may induce bistability of triceps surae motoneurones.