Robert C. Bray

To determine if decreased vascular responsiveness in the medial collateral ligament (MCL) of anterior cruciate ligament transected (ACL-t) rabbit knees is due to pericyte deficiency associated with angiogenesis. Vascular responses to potassium chloride (KCl), phenylephrine, acetylcholine, and sodium nitroprusside (SNP) were evaluated in ACL-t rabbit knees (n = 6) and control knees (n = 5) using laser speckle perfusion imaging. Ligament degeneration was determined by ultrasound imaging. Vascular and pericyte volume were measured using quantitative immunohistochemical volumetric analysis using CD31 and α-smooth muscle actin antibodies with co-localization analysis. Perfusion was increased in the ACL-t rabbits 2.5-fold. Responsiveness to phenylephrine, SNP, and acetylcholine was significantly decreased in the ACL knee while no change in KCl responses was seen. MCL ultrasound imaging revealed decreased collagen organization, increased ligament thickness, and increased water content in the ACL-t MCL. Vascular Volume was increased fourfold in ACL deficient knees, while pericyte volume to endothelial volume was not changed. No difference in CD31 and α-SMA co-localization was found. Blood vessels in the MCL of ACL-t knees do not lack smooth muscle. The MCL vasculature can undergo constrictive response to KCl, but have impaired receptor mediated responses and impaired nitric oxide signaling. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1104–1110, 2014.

Background and Objective Laser speckle perfusion imaging (LSPI) is a minimally invasive optical measure of relative changes in blood flow, providing real‐time, high resolution, two‐dimensional maps of vascular structure. Standard LSI imaging uses a light‐reflective geometry that limits the measurement to a thin surface layer of 0.2–1 mm. The objective of this study was to test a new LSI instrument geometry with the laser source opposed to the image capture plane (light transmissive). Captured light then travels the entire tissue thickness (10–15 mm), sampling much deeper regions of interest than conventional optical imaging techniques. Study Design Reflective‐light (conventional) and transmissive‐light LSI modes were used to measure finger joint blood flow during a timed tourniquet occlusion of the brachial artery in volunteer participants. Results There was greatly increased visibility of vessels underlying the skin in the light‐transmissive mode LSI mode. Established LSI algorithms were shown to still work in the light‐transmissive mode, despite decorrelation due to finite laser coherence length and the light passing through a tissue thickness of 10–15 mm. Conclusion Transmissive LSI can be used to measure blood flow deep (10–15 mm) into tissues. This could be useful for non‐invasive measurements of finger joint synovial blood flow in diagnosing and treating peripheral vascular disorders, such as rheumatoid arthritis. Lasers Surg. Med. 43:21–28, 2011.

Chronic inflammation associated with osteoarthritis (OA) may alter normal vascular responses and contribute to joint degradation. Vascular responses to vasoactive mediators were evaluated in the medial collateral ligament (MCL) of the anterior cruciate ligament (ACL)-deficient knee. Chronic joint instability and progressive OA were induced in rabbit knees by surgical transection of the ACL. Under halothane anesthesia, laser speckle perfusion imaging (LSPI) was used to measure MCL blood flow in unoperated control ( n = 12) and 6-wk ACL-transected knees ( n = 12). ACh, bradykinin, histamine, substance P (SP), and prostaglandin E₂ (PGE₂) were applied to the MCL vasculature in topical boluses of 100 μl (dose range 10⁻¹⁴ to 10⁻⁸ mol). In normal joints, ACh, bradykinin, histamine, and PGE₂ evoked a dilatory response. Substance P caused a biphasic response that was dilatory from 10⁻¹⁴ to 10⁻¹¹ mol and constricting at higher doses. In ACL-deficient knees, ACh, bradykinin, histamine, and SP decreased perfusion, whereas PGE₂ had a biphasic response that decreased perfusion at 10⁻¹⁴ to 10⁻¹¹ mol and was dilatory at higher concentrations. Sodium nitroprusside increased perfusion in resting and phenylephrine-precontracted vessels with no significant differences between ACL-transected and control knees. Femoral artery occlusion and release increased perfusion by 74.3 ± 11.1% in control knees but only by 25.8 ± 4.4% in ACL-deficient knees. The altered responsiveness of the MCL vasculature to these inflammatory mediators may indicate endothelial dysfunction in the MCL, which may contribute to the progression and severity of OA and to the adaptation of the joint in an altered mechanical environment.

Background/purpose: The laser speckle perfusion imaging (LSPI) system is a new, non‐invasive technique for rapidly and reproducibly measuring tissue perfusion. The high resolution and frame rate of the LSPI overcome many of the limitations of traditional laser Doppler imaging techniques. Therefore, LSPI is a useful means for evaluating blood flow in a variety of situations. The present study investigates the ability of the LSPI system to detect temporal changes in blood flow during the healing of cutaneous wounds in a well‐characterized animal model.Methods: Full‐thickness excisional skin wounds (2 × 2 cm) were created on the backs of juvenile female red Duroc pigs. Every week post‐injury, the wounds were measured and photographed, and normalized blood flow values were determined using the LSPI system.Results: Tissue perfusion values were available after complete re‐epithelialization and removal of the eschar, at day 21. At this point, wound blood flow was significantly elevated as compared with the surrounding, uninvolved skin. Wound blood flow declined steadily during healing, and approached normal values by day 35 post‐injury.Conclusion: The kinetics of blood flow during excisional wound healing in the red Duroc model are comparable with that previously observed in laser Doppler imaging of healing human skin wounds and hypertrophic scars. These results therefore confirm that the red Duroc is a good model of human wound healing, and further indicates that the LSPI is an excellent technique for evaluating angiogenesis and neovascularization during healing in this and other models.

This work represents the first clinical data acquired with the endoscopic laser speckle imaging (eLSPI) system, a new diagnostic tool developed for real‐time imaging of tissue blood flow during endoscopic surgical procedures. eLSPI was used to image tissue perfusion in the medial compartment of the knee of five patients requiring arthroscopic knee surgery. The effectiveness of eLSPI as a diagnostic tool was tested by measuring changes in tissue perfusion resultant from tourniquet application, and intra‐articular epinephrine. eLSPI produced real‐time perfusion video images of tissue blood flow in the knee joint. Tourniquet applications produced consistent decreases in mean perfusion index measurements (29.3% ± 5.1% in meniscus; 39.5% ± 8.2% in synovium with an intra patient variability of 6%–9%). A dose‐dependent vasoconstrictive response to the administration of intra‐articular epinephrine was measured, with maximum dose producing a mean decrease in perfusion of 31.0%–9.3% in meniscus and 41.2%–10.9% in synovium. eLSPI consistently detects decreases in articular tissue blood flow resultant from tourniquet inflation or from the administration of increasing concentrations of epinephrine. These are the first in vivo results indicating physiologic changes in articular tissue as a function of two commonly applied practices in endoscopic joint surgery. © 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 24:1650–1659, 2006

Skeletal ligaments are well characterized as mechanical stabilizers of diarthrodial joints. New evidence now suggests that the normal regulation of ligament and joint function may occur through a neural and microvascular “axis,” where the physiology of normal ligaments is influenced by heterogeneous cellular, neural, and microvasculature elements. Within ligament tissue, complex networks of cellular processes linked by gap junctions allow the direct cell-to-cell transfer of signaling molecules, whereas sensory innervation and neurovascular reflexes contribute to motor control and affect ligament mechanical properties. The application of new imaging technologies may assist in determining the functional implications of an integrated neural and microvascular axis. Aging and gender related differences in ligament function are also discussed.

Sympathetic-derived neuropeptide Y (NPY) helps regulate inflammatory responses in injury and disease, is a vasoconstrictor, and stimulates angiogenesis. Rupture of the anterior cruciate ligament (ACL) is a common clinical presentation that results in tissue inflammation, hyperemia, and angiogenesis in the intact medial collateral ligament (MCL). This study is the first to examine the vasoregulatory role of NPY in ACL-deficient knee joints by using the newly developed technique of laser speckle perfusion imaging (LSPI). MCL blood flow was measured in two groups of adult rabbits: unoperated control ( n = 6), and 6-wk ACL transected ( n = 5). Under anesthesia, the MCL was surgically exposed and tissue blood flow was imaged at high resolution using LSPI. NPY was applied to the MCL vasculature in topical boluses of 100 μl (dose range 10⁻¹⁴ to 10⁻⁹ mol), and the α-adrenoceptor agonist phenylephrine was applied in doses of 10⁻¹⁴, 10⁻¹⁰, and 10⁻⁷ mol. In control rabbits, topical administration of NPY or phenylephrine produced dose-dependent vasopressor responses (maximal effect at 10⁻⁹ mol NPY and 10⁻⁷ mol phenylephrine). In ACL-transected knees, there was little or no vasoconstrictive response to NPY at any dose. The response to phenylephrine was significantly reduced compared with control ligaments. Possible causes of the reduced vasoconstrictive response to NPY in the MCL after 6 wk of ACL deficiency include development of tolerance to the peptide due to a prolonged increase in sympathetic nerve activity or change in the distribution or functionality of the NPY Y₁ receptors. Chronic ACL deficiency leads to profound and protracted hyperemia in associated articular tissues. Abrogation of a vasoconstrictor response to both NPY and phenylephrine in the MCL indicates that ACL deficiency induces major changes in the vascular physiological homeostasis.

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.Adult female rabbits were assigned to control, sham operation, ACL hemisection or MCL hemisection groups. Standardized ACL or MCL injuries were surgically induced. About 2, 6 or 16 weeks later, blood flow and vascular volume of the ACL and MCL were measured.The MCL of the rabbit responded to hemisection with a large significant increase in blood flow and a substantial angiogenic response associated with inflammation and scar formation. During subsequent matrix remodelling, blood flow and vascular volume returned towards control values. In contrast, the ACL showed only a 2‐fold increase in vascular volume, no increase in blood flow and atrophied after hemisection. The superior capacity of the MCL to increase its blood supply through angiogenesis and increased flow is essential for ligament healing to occur, and may be the major difference in healing potential between the ACL and MCL. © 2003 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.

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.One‐year‐old female New Zealand white rabbits were assigned to control (n = 12), sham‐operated (n = 12) or PCL transected (2, 6 or 16 weeks, n = 12 per time point) groups. Half of the animals (n = 6 per group) were used for ACL and MCL blood flow determination using coloured microsphere infusion (ml/min/100 g), and half were used for vascular volume determination (given as vascular index, μl/g).In the MCL, PCL transection induced large, significant (4–5‐fold) increases in blood flow (peak at 2 weeks) and vascular index (peak at 6 weeks) compared to sham‐operated animals that returned towards control values by 16 weeks. In contrast, the ACL showed no increase in blood flow in lax joints, and a relatively small (2‐fold) increase in vascular index at 6 weeks only. The wet weight and water content of both the MCL and ACL were significantly increased in PCL‐deficient joints.We conclude that joint laxity (instability) subsequent to loss of the PCL in rabbits impacts the vascular physiology of intact supporting ligaments, inducing both vasomotor and angiogenic responses in the MCL. Changes in wet weight and water content of both the MCL and ACL demonstrate prolonged physiological adaptation of intact structures in lax joints. © 2003 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.