Catherine Leonard

A laser speckle imaging technique for measuring tissue perfusion

Laser Doppler imaging (LDI) has become a standard method for optical measurement of tissue perfusion, but is limited by low resolution and long measurement times. We have developed an analysis technique based on a laser speckle imaging method that generates rapid, high-resolution perfusion images. We have called it laser speckle perfusion imaging (LSPI). This paper investigates LSPI output and compares it to LDI using blood flow models designed to simulate human skin at various levels of pigmentation. Results show that LSPI parameters can be chosen such that the instrumentation exhibits a similar response to changes in red blood cell concentration (0.1%-5%, 200 /spl mu/L/min) and velocity (0-800 /spl mu/L/min, 1% concentration) and, given its higher resolution and quicker response time, could provide a significant advantage over LDI for some applications. Differences were observed in the LDI and LSPI response to tissue optical properties. LDI perfusion values increased with increasing tissue absorption, while LSPI perfusion values showed a slight decrease. This dependence is predictable, owing to the perfusion algorithms specific to each instrument, and, if properly compensated for, should not influence each instruments ability to measure relative changes in tissue perfusion.

Vascular physiology and long-term healing of partial ligament tears

Functional outcomes of anterior cruciate ligament (ACL) injury are generally poorer than those of medial collateral ligament (MCL) tears. Following ligament damage, all phases of ligament healing require an adequate blood supply. We hypothesized that the differences in healing properties of the ACL and MCL would reflect their vascular responses to joint injury. This paper examines the long‐term changes in blood flow and vascular volume of rabbit knee ligaments after direct injury, and under conditions of chronic joint instability induced by section of the posterior cruciate ligament (PCL).

Correlation of healing capacity with vascular response in the anterior cruciate and medial collateral ligaments of the rabbit

In clinical terms, functional recovery after anterior cruciate ligament (ACL) injury is generally poorer than after medial collateral ligament (MCL) injury. In experimental studies of injury, the early phases of ligament healing require an augmented blood supply. We hypothesized that the differences in healing properties of the ACL and MCL would be reflected in the magnitude of their vascular responses to partial injury. This study is the first to quantify and define the time course of changes in blood flow and vascular volume following hemisection of the rabbit ACL and MCL.

Laser Doppler imaging of burn scars: a comparison of wavelength and scanning methods

UNLABELLED Laser Doppler perfusion imaging (LDI) is a useful tool for the early clinical assessment of burn depth and prognostic evaluation of injuries that may require skin grafting. We have evaluated two commercially available laser Doppler imagers for the perfusion measurement of normal and burn scar tissue. METHODS A single wavelength (635 nm), step-wise scanning LDI and a dual wavelength (633 and 780 nm), continuous scanning LDI were used. Twenty patients with hypertrophic burn scars (time since injury: 1 month-8 years) were recruited and the color and elevation of the scar was clinically assessed using a modified Vancouver Burn Scar Scale. Perfusion of each scar region was measured using both imagers. A symmetric contralateral region of unburned skin was also imaged to record baseline perfusion. RESULTS Comparisons of wavelength and scanning technique were made using perfusion values obtained from 22 burn scars. Highly significant positive correlation was observed in all comparisons. In addition, output from both instruments was strongly and significantly correlated with the clinical grading of the scar. SIGNIFICANCE Both LDI scanners perform similar perfusion measurements. The results also indicate that red and near-infrared (NIR) wavelength photons provide similar blood flow information. The faster, continuous scanning method provides a clinical advantage without a significant loss of blood flow information. However, a critical evaluation of both instruments suggests that caution must be exercised when using these optical diagnostic techniques and that some knowledge of light-tissue interaction is required for the proper analysis and interpretation of clinical data.

Reductions in Motoneuronal Neurofilament Synthesis by Successive Axotomies: A Possible Explanation for the Conditioning Lesion Effect on Axon Regeneration

Axons regenerate more rapidly after a test lesion if they received a conditioning lesion. Previous work suggests that the cell body reaction to injury is responsible for this conditioning lesion effect. Here we examined the effects of the second, test lesion on the expression of the major cytoskeletal proteins, tubulin, actin, and neurofilament proteins. Using 2D-SDS-PAGE to separate these cytoskeletal proteins synthesized in the facial nucleus, along with in situ hybridization and RNA blotting to measure corresponding mRNA levels, we found that previous conditioning had little effect on actin or tubulin responses to a test lesion, but resulted in further decrease in neurofilament synthesis. Immunocytochemistry and electron microscopy revealed a greater loss of neurofilaments from the proximal conditioned axons, and axonal shrinkage. We suggest that the reduction in neurofilaments in the proximal axons of conditioned neurons reduces interference with tubulin transport. This may allow more tubulin to be transported more rapidly into the growing axon, to support the faster elongation rate of conditioned axons following a test lesion.

Nerve growth factor improves ligament healing.

Previous work has shown that innervation participates in normal ligament healing. The present study was performed to determine if exogenous nerve growth factor (NGF) would improve the healing of injured ligament by promoting reinnervation, blood flow, and angiogenesis. Two groups of 30 Sprague–Dawley rats underwent unilateral medial collateral ligament transection (MCL). One group was given 10 µg NGF and the other was given PBS via osmotic pump over 7 days after injury. After 7, 14, and 42 days, in vivo blood flow was measured using laser speckle perfusion imaging (LSPI). Morphologic assessments of nerve density, vascularity, and angiogenesis inhibitor production were done in three animals at each time point by immunohistochemical staining for the pan‐neuronal marker PGP9.5, the endothelial marker vWF, and the angiogenesis inhibitor thrombospondin‐2 (TSP‐2). Ligament scar material and structural mechanical properties were assessed in seven rats at each time point. Increased nerve density was promoted by NGF at both 14 and 42 days. Exposure to NGF also led to increased ligament vascularity, as measured by histologic assessment of vWF immunohistochemistry, although LSPI‐measured blood flow was not significantly different from controls. NGF treatment also led to decreased expression of TSP‐2 at 14 days. Mechanical testing revealed that exposure to NGF increased failure load by 40%, ultimate tensile strength by 55%, and stiffness by 30% at 42 days. There were no detectable differences between groups in creep properties. The results suggest that local application of NGF can improve ligament healing by promoting both reinnervation and angiogenesis, and results in scars with enhanced mechanical properties.

Vascular response of the meniscus to injury: effects of immobilization.

Failed meniscal healing may lead to degenerative osteoarthritis of the knee. Healing is thought to be dependent upon an adequate blood supply, yet “normal” vascular changes during healing are not well understood. In this study we have quantified vasoactive and angiogenic responses to medial meniscal injury in a rabbit model under clinically relevant conditions, and related these to histological criteria of healing.

Retrograde changes in transglutaminase activity after peripheral nerve injuries.

It has been previously demonstrated that transglutaminase activity in rat superior cervical ganglion is rapidly and transiently increased minutes after nerve injury. The present series of experiments sought to determine: (1) whether or not similar changes are expressed by other peripheral neuronal systems, and (2) if injury-induced changes in the enzyme activity can be detected along the injured nerve, and if so do they occur in axons or in non-neuronal cells. In the nodose ganglion transglutaminase activity increased (approximately 40%) 48 h after the vagus nerve was crushed 25 mm from the ganglion. In the vagus nerve the activity was transiently increased (approximately 100%) within 1 h, followed by a second increase (approximately 140%) after 3 h. This occurred only in the proximal nerve stump close to the injury site and not in the section of nerve closer to the ganglion. Comparable enzyme activity was found in unoperated vagus nerve and in distal stumps of previously ligated vagus nerves. In dorsal root ganglia no changes were found for up to 24 h after the sciatic nerve was crushed 40 mm from the ganglion. In the facial nucleus a transient increase was observed after the facial nerve was crushed about 14 mm distally with a peak (approximately 300) at 3 days and a decline within 14 days. A second lesion of the facial nerve made 12 days following a conditioning lesion led to a rebound of enzyme activity in the facial nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

Vascular adaptation of intact joint stabilizing structures in the posterior cruciate ligament deficient rabbit knee

Loss of the posterior cruciate ligament (PCL) of the knee has a significant impact on joint stability and biomechanical function. Changes in joint biomechanics may result in mal‐adaptive tissue degeneration and functional alteration of supporting ligaments. This study examines the effects of joint laxity on the vascular physiology of the intact anterior cruciate (ACL) and medial collateral (MCL) ligaments after PCL transection in rabbits.

Allogeneic Bone Marrow Transplant from MRL/MpJ Super-Healer Mice Does Not Improve Articular Cartilage Repair in the C57Bl/6 Strain

Background Articular cartilage has been the focus of multiple strategies to improve its regenerative/ repair capacity. The Murphy Roths Large (MRL/MpJ) “super-healer” mouse demonstrates an unusual enhanced regenerative capacity in many tissues and provides an opportunity to further study endogenous cartilage repair. The objective of this study was to test whether the super-healer phenotype could be transferred from MRL/MpJ to non-healer C57Bl/6 mice by allogeneic bone marrow transplant. Methodology The healing of 2mm ear punches and full thickness cartilage defects was measured 4 and 8 weeks after injury in control C57Bl/6 and MRL/MpJ “super-healer” mice, and in radiation chimeras reconstituted with bone marrow from the other mouse strain. Healing was assessed using ear hole diameter measurement, a 14 point histological scoring scale for the cartilage defect and an adapted version of the Osteoarthritis Research Society International scale for assessment of osteoarthritis in mouse knee joints. Principal Findings Normal and chimeric MRL mice showed significantly better healing of articular cartilage and ear wounds along with less severe signs of osteoarthritis after cartilage injury than the control strain. Contrary to our hypothesis, however, bone marrow transplant from MRL mice did not confer improved healing on the C57Bl/6 chimeras, either in regards to ear wound healing or cartilage repair. Conclusion and Significance The elusive cellular basis for the MRL regenerative phenotype still requires additional study and may possibly be dependent on additional cell types external to the bone marrow.