A reproducible method for biochemical, histological and functional assessment of the effects of ischaemia-reperfusion syndrome in the lower limbs.

Current methodology described to mimic lower limb ischaemia-reperfusion injury (LL-IRI) does not accurately define the procedures and pressures exerted to induce and maintain ischaemia. In this piece of work, we propose a well-defined and detailed rat model that simulates the conditions established in clinical practice guidelines for tourniquet application and allows us to test treatments that aim to prevent/reduce LL-IRI. Eighty-six male WAG/RijHsd rats were subjected to hind limb IRI (LL-IRI), using a mechanical system applying a 1 kg tension to induce and maintain ischemia for 2 or 3 h, and assessed the damage caused by reperfusion at biochemical and muscular levels at different time points. At the biochemical level, both 2 and 3 h of ischemia induced changes (except for electrolyte levels); 3 h of ischemia induced greater changes in specific markers of muscular damage: creatine kinase (CK) and lactate dehydrogenase (LDH). At the histopathological level, 3 h of ischemia and 24 h of reperfusion was associated with an increase in hind limb girth, cross-sectional area, and weight and presence of neutrophils, as well as histological damage in more than 60% of muscle fibres. Our model allows to reliably reproduce the damage associated with the use of a pneumatic tourniquet. CK and LDH, as well as measures of tissue damage, allow to define and characterize the response to LL-IRI-related damage. A period of 3 h of ischemia followed by 3 h of reperfusion caused only local damage but showed greater sensitivity to detect differences in future studies on prophylactic treatments against LL-IRI.

Assessment of the Efficacy and Safety of a Novel, Low-Cost, Junctional Tourniquet in a Porcine Model of Hemorrhagic Shock.

INTRODUCTION: Commercially available junctional tourniquets (JTQs) have several drawbacks. We developed a low-cost, compact, easy to apply JTQ. The aim of this study was to assess the tourniquets' safety and efficacy in a swine model of controlled hemorrhage. MATERIALS AND METHODS: Five pigs were subjected to controlled bleeding of 35% of their blood volume. Subsequently, the JTQ was applied to the inguinal area for 180 minutes. Afterwards, the tourniquet was removed for additional 60 minutes of follow up. During the study, blood flow to both hind limbs and blood samples for tissue damage markers were repeatedly assessed. Following sacrifice, injury to both inguinal areas was evaluated microscopically and macroscopically. RESULTS: Angiography demonstrated complete occlusion of femoral artery flow, which was restored following removal of the tourniquet. No gross signs of tissue damage were noticed. Histological analysis revealed mild necrosis and infiltration of inflammatory cells. Blood tests showed a mild increase in potassium and lactic acid levels throughout the protocol. CONCLUSIONS: The tourniquet achieved effective arterial occlusion with minimal tissue damage, similar to reports of other JTQs. Subjected to further human trials, the tourniquet might be a suitable candidate for widespread frontline deployment because of its versatility, compactness, and affordable design.

Variations in Surgical Procedures for Inducing Hind Limb Ischemia in Mice and the Impact of These Variations on Neovascularization Assessment.

Mouse hind limb ischemia is the most common used preclinical model for peripheral arterial disease and critical limb ischemia. This model is used to investigate the mechanisms of neovascularization and to develop new therapeutic agents. The literature shows many variations in the model, including the method of occlusion, the number of occlusions, and the position at which the occlusions are made to induce hind limb ischemia. Furthermore, predefined end points and the histopathological and radiological analysis vary. These differences hamper the correlation of results between different studies. In this review, variations in surgical methods of inducing hind limb ischemia in mice are described, and the consequences of these variations on perfusion restoration and vascular remodeling are discussed. This study aims at providing the reader with a comprehensive overview of the methods so far described, and proposing uniformity in research of hind limb ischemia in a mouse model.

Preclinical quantitative in-vivo assessment of skin tissue vascularity in radiation-induced fibrosis with optical coherence tomography.

Radiation therapy (RT) is widely and effectively used for cancer treatment but can also cause deleterious side effects, such as a late-toxicity complication called radiation-induced fibrosis (RIF). Accurate diagnosis of RIF requires analysis of histological sections to assess extracellular matrix infiltration. This is invasive, prone to sampling limitations, and thus rarely used; instead, current practice relies on subjective clinical surrogates, including visual observation, palpation, and patient symptomatology questionnaires. This preclinical study demonstrates that functional optical coherence tomography (OCT) is a useful tool for objective noninvasive in-vivo assessment and quantification of fibrosis-associated microvascular changes in tissue. Data were collected from murine hind limbs 6 months after 40-Gy single-dose irradiation and compared with nonirradiated contralateral tissues of the same animals. OCT-derived vascular density and average vessel diameter metrics were compared to quantitative vascular analysis of stained histological slides. Results indicate that RIF manifests significant microvascular changes at this time point posttreatment. Abnormal microvascular changes visualized by OCT in this preclinical setting suggest the potential of this label-free high-resolution noninvasive functional imaging methodology for RIF diagnosis and assessment in the context of clinical RT.

Simvastatin augments revascularization and reperfusion in a murine model of hind limb ischemia - Multimodal imaging assessment.

INTRODUCTION: Peripheral artery disease can lead to severe disability and limb loss. Therapeutic strategies focussing on macrovascular repair have shown benefit but have not significantly reduced amputation rates in progressive PAD. Proangiogenic small molecule therapies may substantially improve vascularisation in limb ischemia. The purpose of the current study was to assess the proangiogenic effects of simvastatin in a murine model of hind limb ischemia using longitudinal multimodal imaging. METHODS: Mice underwent surgical intervention to induce hind limb ischemia, and were treated with simvastatin orally for 28days. Neovascularisation was assessed using 99mTc-RGD SPECT imaging, and macrovascular volume was assessed by quantitative time of flight MRI. At each imaging time point, VEGF expression and capillary vessel density were quantified using immunohistochemical analysis. RESULTS: Simvastatin significantly increased 99mTc-RGD retention in the ischemic hind limb by day 3 post-surgery, with maximal retention at day 8. Vascular volume was significantly increased in the ischemic hind limb of simvastatin treated animals, but only by day 22. Immunohistochemical analysis shows that simvastatin significantly augmented tissue VEGF expression from day 8 with increase in capillary density (CD31+) from day 14. CONCLUSIONS: Early assessment of proangiogenic therapy efficacy can be identified using 99mTc-RGD SPECT, which displays significant increases in retention before macrovascular volume changes are measureable with MRI. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: Simvastatin offers an effective proangiogenic therapy as an adjunct for management of limb ischemia. Simvastatin induces integrin expression and vascular remodeling leading to neovascularisation and improved perfusion.

Therapeutic assessment of mesenchymal stem cells delivered within a PEGylated fibrin gel following an ischemic injury.

The intent of the current study was to investigate the therapeutic contribution of MSCs to vascular regeneration and functional recovery of ischemic tissue. We used a rodent hind limb ischemia model and intramuscularly delivered MSCs within a PEGylated fibrin gel matrix. Within this model, we demonstrated that MSC therapy, when delivered in PEGylated fibrin, results in significantly higher mature blood vessel formation, which allows for greater functional recovery of skeletal muscle tissue as assessed using force production measurements. We observed initial signs of vascular repair at early time points when MSCs were delivered without PEGylated fibrin, but this did not persist or lead to recovery of the tissue in the long-term. Furthermore, animals which were treated with PEGylated fibrin alone exhibited a greater number of mature blood vessels, but they did not arterialize and did not show improvements in force production. These results demonstrate that revascularization of ischemic tissue may be a necessary but not sufficient step to complete functional repair of the injured tissue. This work has implications on stem cell therapies for ischemic diseases and also potentially on how such therapies are evaluated.

[¹¹C]acetate and PET/CT assessment of muscle activation in rat studies.

PURPOSE: The purpose of the present study is to apply kinetic analysis to investigate exercise-related changes in the metabolism of the skeletal muscle of the rat hindlimb by [[Formula: see text]]acetate positron emission tomography and computed tomography (PET/CT). METHODS: Contractions were induced in Wistar rats' left hindlimb by electrostimulation of the Vastus Lateralis muscle motor point. After 15 min of muscle contractions, [[Formula: see text]]acetate was injected and PET/CT of both hindlimbs was acquired. The resting hindlimb was used as a control reference. The kinetic parameters [Formula: see text] and [Formula: see text] were calculated for the target muscles (exercised and control) and correlated with the corresponding standardized uptake values (SUVs). The ratio between each kinetic parameter values and the SUV extracted for the exercised muscle and the muscle at rest was computed ([Formula: see text] and [Formula: see text], respectively). RESULTS: Kinetic analysis quantitatively confirmed that net tracer uptake ([Formula: see text]) and washout ([Formula: see text]) were significantly higher in exercised muscles ([Formula: see text] for exercised muscles vs. [Formula: see text] for resting muscles, [Formula: see text]; [Formula: see text] for exercised muscle vs. [Formula: see text] for resting muscle, [Formula: see text]). On the other hand, SUV was not significantly different between active and inactive muscles ([Formula: see text] for exercised muscles vs. [Formula: see text] for resting muscles). Linear regression analysis revealed a good correlation ([Formula: see text]) between net tracer uptake ratio ([Formula: see text]) and the SUV ratio [Formula: see text]). A lower correlation was found between the net tracer washout ratio ([Formula: see text]) and the SUV ratio ([Formula: see text]). CONCLUSION: The present study showed that kinetic modelling can detect changes between active and inactive skeletal muscles with a higher sensitivity with respect to the SUV, when performed with [[Formula: see text]]acetate PET/CT.

Spatial vascular volume fraction imaging for quantitative assessment of angiogenesis.

PURPOSE: The purpose of this study is to set up a quantitative imaging strategy for therapeutic angiogenesis and monitoring the spatial distribution and survival of the transplanted mesenchymal stem cells (MSCs) in vivo simultaneously. PROCEDURES: Mouse adipose-derived MSCs (AD-MSCs) were isolated from firefly luciferase and enhanced green fluorescent protein positive transgenic mice, and implanted intramuscularly into hindlimbs of C57BL/6 mice. Serial spatial vascular volume fraction (SVVF) imaging was performed to quantitatively assess angiogenesis by calculating the spatially explicit vascular volume. The hybrid microcomputed tomography angiography/bioluminescence tomography (micro-CTA/BLT) was used to track the fate of AD-MSCs in vivo. Laser Doppler perfusion imaging (LDPI) was used to evaluate blood perfusion. Ex vivo conventional methods were performed to cross-validate the therapeutic angiogenesis. RESULTS: There was a linear correlation relation between the cell number and Fluc/Fluc protein signal intensity in AD-MSCs via BLT. LDPI showed improved perfusion rate in mice treated with cytotherapy, compared to control mice. Furthermore, angiogenesis assessed by SVVF was 10.67 ± 0.41 %, 13.99 ± 0.28 %, and 23.50 ± 1.23 % on days 7, 14, and 28 post-transplantation of AD-MSCs, respectively. Vascular densities of the longitudinally monitored ischemic hindlimbs were significantly higher than those at early time points and controls, which was also confirmed by vascular corrosion casting, scanning electron microscopic imaging, and histological analysis. CONCLUSIONS: Hybrid high-resolution micro-CTA/BLT enabled monitoring and quantitative assessment of cytotherapeutically induced angiogenesis in vivo.

Assessment of protective effects of methylprednisolone and pheniramine maleate on reperfusion injury in kidney after distant organ ischemia: a rat model.

BACKGROUND: Ischemia/reperfusion (I/R) injury of tissues is a common problem that cardiovascular surgeons are faced with. Suppression of inflammation, which plays an important role in the pathogenesis of I/R injury, may reduce this damage. The aim of this study is to investigate the protective effects of methylprednisolone (MP)--a potent anti-inflammatory agent--and pheniramine maleate (FM)--an antihistamine that also has some anti-inflammatory effects--on reperfusion injury of kidneys developing after ischemia of the left lower extremity of rats. METHODS: Twenty-eight randomly selected male Sprague-Dawley rats weighing 320 to 370 g were divided into four groups, each consisting of seven rats. Group 1 was the control group. Group 2 was the sham group. Rats in group 3 were subjected to I/R and given FM, and rats in group 4 were subjected to I/R and given MP. A tourniquet was applied at the level of the left groin to subjects in group 2 after induction of anesthesia. One hour of ischemia was performed, and no drug was administered. In group 3, half of a total dose of 10 mg/kg FM was administered before ischemia, and the remaining half was given intraperitoneally before reperfusion. In group 4, subjects received a single dose of 50 mg/kg MP intraperitoneally in the 30th minute of ischemia. Kidneys of all subjects were removed after 24 hours. Extracted tissues were investigated regarding histological and biochemical parameters. RESULTS: Malondialdehyde--the end product of lipid peroxidation as an important indicator of I/R injury--levels were significantly lower in group 3 than in group 2 (P < 0.05). Malondialdehyde levels were also lower in group 4 than in group 2, but this difference was insignificant (P > 0.05). Superoxide dismutase and glutathione peroxidase enzyme activities were found to be significantly higher in group 3 than in group 2 (P < 0.05). However, there was no difference between group 4 and group 2 in terms of these activities. Histological examination demonstrated that both MP and FM had protective effects against I/R injury, but this effect was more potent for FM than for MP. CONCLUSIONS: FM has a protective effect against reperfusion injury in rat kidney after distant organ ischemia.

Assessment of gait dynamics in rats submitted to limb ischemia / Avaliação da dinâmica da marcha em ratos submetidos à isquemia de membro

PURPOSE: To describe a method for the assessment of gait dynamics in rats submitted to limb ischemia. METHODS: Twenty-four male Wistar rats (150-160g) were used. Twelve animals were submitted to limb ischemia by ligation of the common left iliac artery (ischemic group: n = 12); and a sham-operated group was used as control (n=12). After a recovery period of 6 weeks, gait dynamics was assessed by counting the complete footprints and the number of hindlimb-floor contacts during a treadmill test for five minutes at a speed of 12 m.min-1 and angulation of 15°. The number of contacts of the left hindlimb was divided by the right hindlimb values (LRR) for group comparisons. Ischemic disability was quantified by comparing the area under curve (AUC) created by plotting each contact versus time for each hindlimb. The left hindlimb ischemic disability index (LHDI), which was compared between groups, was defined by the formula: LHDI = (1- AUC left / AUC right) x 100. RESULTS: Surgery was well tolerated by all animals. Rats did not suffer tissue loss or ulcerations. Complete footprint LRR was 0.3 ± 0.08 for the ischemic group and 1.3 ± 0.9 for controls (p=0.0043). Number of contacts LLR was 0.5 ± 0.2 for the ischemic group and 1.0 ± 0.1 for the control group (p=0.0051). LHDI was 56.83 ± 10.67 for the ischemic group and 2.50 ± 13.10 for the control group (P = 0.031). CONCLUSION: Assessment of gait dynamics in rats submitted to limb ischemia could be done by footprint analysis and hindlimb contact recording during a treadmill test.

OBJETIVO: Descrever um método para avaliar a dinâmica da marcha em ratos submetidos à isquemia de membro pélvico. MÉTODOS: Vinte e quatro ratos Wistar machos (150-160g) foram utilizados neste estudo experimental. Doze animais foram submetidos à isquemia de membro pélvico por meio da ligadura da artéria ilíaca comum esquerda (grupo isquêmico: n=12); e doze animais foram submetidos à cirurgia simulada e usados como controle (grupo controle: n=12). Após seis semanas de recuperação, foi realizada avaliação da dinâmica da marcha por meio da contagem de impressões plantares e da contagem de contatos pata-solo durante teste com esteira durante cinco minutos, velocidade 12 m.min-1 e angulação de 15°. Os valores do número de contatos do membro pélvico esquerdo foram divididos pelos do membro pélvico direito (razão esquerda-direita - LRR) para comparação entre os grupos. A quantificação da incapacitação isquêmica foi feita comparando a área sob a curva (AUC) da representação gráfica dos contatos versus tempo para cada membro pélvico. O índice de incapacitação isquêmica do membro pélvico esquerdo (LHDI), que foi comparado entre os grupos, foi definido pela fórmula: LHDI = (1- AUC esquerda / AUC direito) x 100. RESULTADOS: A cirurgia foi bem tolerada por todos os animais. Nenhum rato apresentou necrose tecidual ou ulceração. A LRR das impressões plantares completas foi 0,3 ± 0,08 no grupo isquêmico e 1,3 ± 0,9 no grupo controle (p=0,0043). A LRR do número de contatos foi 0,5 ± 0,2 no grupo isquêmico e 1,0 ± 0,1 no grupo controle (p=0,0051). O LHDI foi 56,83 ± 10,67 no grupo isquêmico e 2,50 ± 13,10 no grupo controle (p=0,031). CONCLUSÃO: Avaliação da dinâmica da marcha em ratos submetidos à isquemia de membro pélvico pôde ser feita por meio da contagem de impressões plantares e da contagem de contatos pata-solo durante teste com esteira.

Assessment of gait dynamics in rats submitted to limb ischemia.

PURPOSE: To describe a method for the assessment of gait dynamics in rats submitted to limb ischemia. METHODS: Twenty-four male Wistar rats (150-160g) were used. Twelve animals were submitted to limb ischemia by ligation of the common left iliac artery (ischemic group: n = 12); and a sham-operated group was used as control (n=12). After a recovery period of 6 weeks, gait dynamics was assessed by counting the complete footprints and the number of hindlimb-floor contacts during a treadmill test for five minutes at a speed of 12 m.min(-1) and angulation of 15°. The number of contacts of the left hindlimb was divided by the right hindlimb values (LRR) for group comparisons. Ischemic disability was quantified by comparing the area under curve (AUC) created by plotting each contact versus time for each hindlimb. The left hindlimb ischemic disability index (LHDI), which was compared between groups, was defined by the formula: LHDI = (1- AUC (left) / AUC (right)) x 100. RESULTS: Surgery was well tolerated by all animals. Rats did not suffer tissue loss or ulcerations. Complete footprint LRR was 0.3 ± 0.08 for the ischemic group and 1.3 ± 0.9 for controls (p=0.0043). Number of contacts LLR was 0.5 ± 0.2 for the ischemic group and 1.0 ± 0.1 for the control group (p=0.0051). LHDI was 56.83 ± 10.67 for the ischemic group and 2.50 ± 13.10 for the control group (P = 0.031). CONCLUSION: Assessment of gait dynamics in rats submitted to limb ischemia could be done by footprint analysis and hindlimb contact recording during a treadmill test.

Contrast sonography enables noninvasive and quantitative assessment of neovascularization after stem cell transplantation.

Stem cell transplantation is one of the attractive therapeutic strategies for the treatment of hindlimb ischemia. However, few studies have quantitatively assessed perfusion noninvasively in deep tissues after cell transplantation. In this study, we examined the feasibility of contrast sonography for the assessment of perfusion after bone marrow-derived mesenchymal stem cell (MSC) transplantation by using a rat unilateral hindlimb ischemia model. The quantitative parameters derived from contrast sonography were compared with the colored microspheres-derived blood flow and the capillary density. Nine rats were assigned each to a control (saline injection) or a treated (MSC transplantation) group. Video intensity vs. pulsing interval plots were acquired with ultraharmonic imaging of SONOS5500 during IV infusion of Levovist. The left-to-right ratio of hindlimb blood volume (A-ratio), microbubble velocity (beta-ratio) and hindlimb blood flow (Abeta-ratio) were calculated. The MS-ratio, the ratio of the left to the right hindlimb blood flow determined using colored microspheres, was also calculated. Although A-ratio did not change, beta- and Abeta-ratio in the treated group were significantly higher than those in the control group. In addition, MS-ratio and capillary density in the treated group were significantly higher than those in the control group. Compared with A- and Abeta-ratio, beta-ratio had the highest correlation with MS-ratio and capillary density (vs. MS-ratio: r = 0.66, p < 0.01; vs. capillary density: r = 0.52, p < 0.05). The results of our study imply that the contrast sonography-derived beta-ratio is a useful parameter that reflects the perfusion after cell transplantation in ischemic hindlimb.

Assessment of ischemia-induced microvascular remodeling using contrast-enhanced ultrasound vascular anatomic mapping.

Our aim was to apply novel contrast-enhanced ultrasound (CEU) techniques to characterize remodeling in different vascular compartments during ischemia-mediated angiogenesis. Hind limb ischemia was produced by ligation of an external iliac artery in 60 rats, half of which were treated with intramuscular fibroblast growth factor (FGF)-2 (5 microg). The proximal adductor muscles of the ischemic and control hind limb were studied immediately after ligation and at days 4, 7, or 14. Low-power maximum intensity projection imaging was performed to assess large intramuscular vessels to the fourth branch order. CEU data were analyzed to measure capillary perfusion and functional noncapillary microvascular blood volume. Resting capillary perfusion was reduced by 30% after arterial ligation and recovered earlier in FGF-2-treated versus nontreated rats (day 4 vs. 14). Changes in perfusion were temporally related to expansion of noncapillary microvascular blood volume on CEU, which was associated with an arteriogenic response on histology. Expansion of and organization (fractal distribution) of large collateral vessels occurred gradually over 2 weeks and was slightly more rapid with FGF-2 treatment. We conclude that CEU can separately assess collateral development, more distal arteriogenesis, and secondary changes in capillary perfusion that occur differentially with ischemia and growth factor therapy.

Contrast-enhanced triggered harmonic sonography for assessment of periarticular hemodynamic changes in experimental arthritis.

BACKGROUND: Objective quantification is critical for assessment of functional sonography in inflammatory arthritis. To create a microbubble contrast-enhanced image of vessels that lie below the resolution of a standard US system, a technique is required that detects preferentially the contrast agent echo, rejecting that from background tissue: harmonic imaging. OBJECTIVES: To investigate the ability of contrast-enhanced triggered harmonic sonography (CETHS) to evaluate periarticular hemodynamic changes over the course of experimental arthritis and to discriminate presence and absence of arthritis based on measurement values obtained at specific time-points. MATERIALS AND METHODS: Arthritis was induced in rabbits knees by intra-articular injection of serum bovine albumin, which acted as an antigen. A total of 11 rabbits (8 with unilateral arthritis and 3 control animals) were imaged at 0, 1, 7, 14, 21 and 28 days of antigen-induced arthritis and euthanized at 28 days. A continuous infusion protocol was performed (triggering times 30.0, 20.0, 10.0, 5.0, 2.0, 1.0, and 0.5 s). Hemodynamic indices of synovial microvasculature (vascular volume, mean velocity and flow rate) were obtained and compared with clinical, laboratory, and histological surrogate markers. RESULTS: Although interval CETHS changes were noted for flow rate (P=0.007) and vascular volume (P=0.003) ratios in albumin-injected knees, no significant differences in ratios were identified over time between albumin-injected and non-injected knees for flow rate (P=0.52), vascular volume (P=0.23) and mean velocity (P=0.19). Flow rate most accurately differentiated between presence and absence of arthritis according to clinical measurements in early (day 1) arthritis, and mean velocity in mid-term arthritis (day 14; both P=0.02). CONCLUSION: Although the measurement properties of CETHS indices were poor in the evaluation of hemodynamic differences over time in albumin-injected knees compared with non-injected knees, they enabled discrimination between presence and absence of arthritis at specific time-points in different stages.

Assessment of endothelium-dependent vasodilation in equine digital resistance vessels.

Haemodynamic disturbances leading to ischaemia and reperfusion injury of the digit are thought to be involved in the pathophysiology of acute equine laminitis. Identification of physiological regulators of blood flow through the equine digit is important in identifying factors, which may predispose animals to laminitis. A method was developed to assess endothelium-dependent responses of the isolated Krebs-perfused equine digit by co-administration of 5-hydroxytryptamine (5-HT) with vasodilator agents, carbachol (CCh), bradykinin (BK) and substance P (SP). Bolus co-administration of CCh (0.02-2 micromol), BK and SP (0.02-0.2 nmol), caused inhibition of the 5-HT pressor response by 50-60%. The vasodilator responses were abolished by the detergent, CHAPS, indicating endothelium dependency; whereas vasoconstrictor responses to 5-HT were potentiated. CCh-induced relaxation was significantly reduced by the nitric oxide synthase inhibitor L-NAME (79.7 +/- 3.4% inhibition), whereas a large proportion of BK and SP-induced relaxation remained (34.1 +/- 6.3% and 33.6 +/- 5.3% inhibition). L-NAME potentiated vasoconstrictor responses to 5-HT. In conclusion, this study demonstrates that endothelium-derived NO modulates the response to vasoconstrictors such as 5-HT and is likely to be an important regulator of blood flow in the digital resistance vascular bed. Other factor(s) released by the endothelium are also important in regulating blood flow, whose identity remains to be established.

DTI-based assessment of ischemia-reperfusion in mouse skeletal muscle.

Diffusion tensor imaging (DTI) is frequently applied to characterize the microscopic geometrical properties of tissue. To establish whether and how diffusion MRI responds to transient ischemia of skeletal muscle, we studied the effects of ischemia and reperfusion using DTI and T2-weighted MRI before and during ischemia and up to 24 hr after reperfusion. Ischemia was induced by 50 min of hindlimb occlusion with or without dorsal flexor stimulation. During ischemia the apparent diffusion coefficient (ADC) tended to decrease (up to 15%), whereas the fractional anisotropy (FA) and T2 showed a varied response depending on the protocol and muscle type. During reperfusion the ADC and T2 initially increased and subsequently renormalized for the occlusion protocol. For the occlusion plus stimulation (OS) protocol, the FA was decreased by 13% and the ADC and T2 were increased by 20% and 57%, respectively, after 24 hr in the stimulated muscle complex. In the latter tissue the three DTI eigenvalues gradually increased upon reperfusion. The smallest eigenvalue (lambda3) showed the largest relative increase. Changes in DTI indices in the reperfusion phases followed a similar time course as the changes in T2. The changes in MR indices after 24 hr correlated with the tissue damage quantified with histology. The highest correlation was observed for lambda3 (R2 = 0.81). This study shows that DTI can be used to assess ischemia-induced damage to skeletal muscle.

Comparison of six methods for the assessment of ischemia-reperfusion injury in skeletal muscle following composite tissue allotransplantation.

Ischemia/reperfusion (I/R) injury is one of the factors determining tissue survival in replantation and transplantation surgery. However, more than 20 methods have been used to evaluate I/R injury in muscle. The aim of this study was to examine I/R injury in muscle tissue in a model of composite tissue allotransplantation (rat hindlimb transplantation), based on the analyses of six parameters: nitroblue tetrazolium staining (NBT); histology of the anterior tibial and extensor digitorum muscle; wet-to-dry weight ratio; serum potassium; and serum creatine kinase (CK)), in order to identify the most practicable and reliable outcome parameter. Results demonstrated that NBT staining and the wet/dry weight ratio are reliable tools for outcome measurement. The wet/dry weight ratio is the easiest to perform and the authors consider it to be useful for screening purposes. Histologic assessment shows areas of necrosis, but is not a reliable method for semi-quantitative evaluation. Serum potassium and CK levels were higher following transplantation, but they cannot be recommended for assessment purposes, as no significant correlation with other parameters was seen. These findings help further researchers in their selection of reliable outcome parameters to measure I/R injury in skeletal muscle.

In vivo assessment of absolute perfusion in the murine skeletal muscle with spin labeling MRI. Magnetic resonance imaging.

PURPOSE: To assess absolute perfusion in the skeletal muscle of mice in vivo with spin labeling magnetic resonance imaging (MRI) under normal and stress conditions. MATERIALS AND METHODS: Absolute perfusion in the skeletal muscle of 27 C57BL/6 mice was assessed in vivo non-invasively by spin labeling MRI at 7.05 T. This technique was based on the acquisition of T1 maps with global and slice-selective spin inversion in separate acquisitions. T1 mapping was performed by inversion recovery snapshot fast low angle shot imaging. To guarantee proper spin inversion within the whole mouse, a dedicated radiofrequency (RF) coil combination was constructed. A birdcage resonator was used for transmission, while detection of the MRI signal was achieved by a surface coil. RESULTS: Basal perfusion in the hindlimbs was determined to be 94 +/- 10 mL (100 g x minute)(-1) (mean +/- standard error of the mean [SEM], N = 27). This value is in good agreement with perfusion values determined by invasive techniques such as microspheres. A subgroup of six animals received a constant dose of 4 mg (kg x minute)(-1) of the vasodilator adenosine by an intraperitoneal catheter. In this case, perfusion was significantly increased to 179 +/- 56 mL (100 g x minute)(-1) (mean +/- SEM, N = 6, P < 0.02). Mean basal perfusion in this subgroup was 96 +/- 26 mL (100 g x minute)(-1). CONCLUSION: Spin labeling MRI is a well-suited technique for the in vivo assessment of absolute perfusion in the murine skeletal muscle.

Assessment of tissue blood flow following small artery welding with an intraluminal dissolvable stent.

Using the technique of radioactive 51Cr-labeled biological microspheres, this study evaluated arterial blood flow following small vessel anastomosis by CO2 laser welding and a dissolvable stent in the lumen. A total of 30 Sprague-Dawley rats were divided into two groups. Group A: 11 rats had their femoral arteries ligated on one side. The contralateral side served as a control, with the artery transected and repaired using conventional microsuturing. Group B: 19 rats had their femoral arteries transected and repaired using CO2 laser welding and an intraluminal dissolvable stent technique. The contralateral side was again used as a control using conventional microsuturing. At 1 hr postoperatively, 51Cr-labeled biological microspheres were injected centripetally into the left common carotid artery and the legs and thighs immediately harvested for measurement of radioactivity. All repaired arteries were patent (30/30 in the microsuturing group and 19/19 in the stented welding group), with no detectable stenosis or dilation at the repaired site. Statistical analysis showed that tissue radioactivity (cpm/g) in the ligated group (3,972 +/- 384 in thighs and 3,142 +/- 742 in legs) was significantly lower than in the microsuturing group (7,132 +/- 1,723 in thighs and 6,557 +/- 1,469 in legs) (P < 0.01). In the ligated group, a significant reduction of blood flow was seen in the legs when compared with the thighs (P < 0.05). There was no significant difference in radioactivity when comparing the microsuturing control with the stented welding group, in both thighs (7,064 +/- 2,599 and 7,006 +/- 2,406, respectively; P > 0.05) and legs (6,386 +/- 1,703 and 6,288 +/- 1,757, respectively; P > 0.05). This study provided evidence that the dissolvable stent placed intraluminally does not impair blood circulation and that when coupled with CO2 laser welding offers a high-quality alternative to conventional small vessel anastomosis.

Assessment of the effects of endothelin-1 and magnesium sulphate on regional blood flows in conscious rats, by the coloured microsphere reference technique.

There is evidence to suggest that magnesium (Mg2+) is beneficial in the treatment of a number of conditions, including pre-eclampsia and acute myocardial infarction. The mode of action of Mg2+ in these conditions is not clear, although the vasodilator properties of Mg2+ are well documented both in vitro and in vivo. Previously, we demonstrated that i.v. infusion of magnesium sulphate (MgSO4) alone, or in the presence of vasoconstrictors, caused increases in flow and conductance in the common carotid, internal carotid and hindquarters vascular beds, in conscious rats. Therefore, the objective of the present study was to investigate the regional and subregional changes in haemodynamics in response to the vasoconstrictor peptide endothelin-1 (ET-1) and MgSO4 in more detail, using the coloured microsphere reference technique. Infusion of ET-1 and MgSO4 had similar effects on heart rate and mean arterial pressure as in our previous study. Infusion of ET-1 caused a rise in mean arterial pressure and a fall in heart rate, and infusion of MgSO4 returned mean arterial pressure to control levels with no effect on heart rate. The responses to MgSO4 in the presence of ET-1 showed considerable regional heterogeneity with blood flow increasing (e.g. skeletal muscle), decreasing (e.g. stomach) or not changing (e.g. kidney). Of particular interest was the finding that MgSO4 caused increases in flow in the cerebral and coronary vascular beds. This, and our previous studies, have shown that MgSO4 can reverse vasoconstriction in a number of vascular beds, and indicate that this compound may have therapeutic benefit in conditions associated with vasospasm.