Pig Hind Limb Research Articles

Effect of temporary freezing on postmortem protein degradation patterns

BackgroundA precise determination of time since death plays a major role in forensic routine. Currently available techniques for estimating the postmortem interval (PMI) are restricted to specific time periods or cannot be applied for individual case-specific reasons. During recent years, it has been repeatedly demonstrated that Western blot analysis of postmortem muscle protein degradation can substantially contribute to overcome these limitations in cases with different background. Enabling to delimit time points at which certain marker proteins undergo distinct degradation events, the method has become a reasonable new tool for PMI delimitation under various forensic scenarios. However, additional research is yet required to improve our understanding of protein decomposition and how it is affected by intrinsic and extrinsic factors.Since there are temperature limits for proteolysis, and investigators are confronted with frozen corpses, investigation of the effects of freezing and thawing on postmortem protein decomposition in the muscle tissue is an important objective to firmly establish the new method. It is also important because freezing is often the only practical means to intermittently preserve tissue samples from both true cases and animal model research.MethodsSets of dismembered pig hind limbs, either freshly detached non-frozen, or thawed after 4 months of freeze-storage (n = 6 each), were left to decompose under controlled conditions at 30 °C for 7 days and 10 days, respectively. Samples of the M. biceps femoris were regularly collected at predefined time points. All samples were processed via SDS-PAGE and Western blotting to identify the degradation patterns of previously characterized muscle proteins.ResultsWestern blots show that the proteins degrade predictably over time in precise patterns that are largely unaffected by the freeze-and-thaw process. Investigated proteins showed complete degradation of the native protein band, partly giving rise to degradation products present in distinct time phases of the decomposition process.ConclusionThis study provides substantial new information from a porcine model to assess the degree of bias that freezing and thawing induces on postmortem degradation of skeletal muscle proteins. Results support that a freeze–thaw cycle with prolonged storage in frozen state has no significant impact on the decomposition behavior. This will help to equip the protein degradation–based method for PMI determination with a robust applicability in the normal forensic setting.

Abstract 13133: Heterogeneous Uptake of [ 99m Tc]microspheres and 201 Tl in Skeletal Muscles in Porcine Model of Hindlimb Ischemia

Introduction: There is a need for quantitative methods to evaluate skeletal muscle blood flow in PAD patients. We assessed skeletal muscle blood flow heterogeneity at rest or vasodilator stress using 99m Tc-microspheres (HSA), that do not cross the capillaries, and 201 Tl, a freely diffusible radiotracer, in a pig hindlimb ischemia model. Methods: Following acute right femoral artery ligation, pigs (n=8) were injected with either 201 Tl or HSA at rest (n=2 per tracer) or under adenosine-stress (n=2 per tracer). Ex vivo SPECT/CT imaging was performed on excised ischemic (I) and non-ischemic (NI) gastrocnemius (GM) and soleus (SM) muscles. SPECT images were analyzed for the ischemic/nonischemic (I/NI) activity ratio, tracer heterogeneity as indicated by the coefficient of variance (CV) of signal intensity and image intensity histograms. Results: The rest 201 Tl I/N ratio for SM was higher (0.86±0.08) compared to stress (0.64±0.16), with no difference in GM. In contrast, the I/N ratio for HSA was near normal in both muscle groups at rest (GM: 0.80±0.21, SM: 1.06±0.45) although decreased during stress (GM: 0.70±0.04, SM: 0.71±0.12). Rest 201 Tl displayed uniformly low CV in both I (GM: 0.43, SM: 0.43) and NI muscles (GM: 0.37, SM: 0.43). Corresponding CV for stress 201 Tl were similar (I - GM: 0.41, SM: 0.42; NI - GM: 0.41, SM: 0.46). Rest HSA CV were higher in I (GM: 0.92, SM: 1.03) and NI (GM: 0.67, SM: 0.69) muscle, although there was greater heterogeneity in the I muscles assessed with HSA. Stress HSA CV decreased in both I (GM: 0.60, SM: 0.73) and NI (GM: 0.52, SM: 0.51). Figure displays representative count histograms. Conclusions: 201 Tl displayed less heterogeneity than HSA. 201 Tl appears to be more sensitive at detecting reduced blood flow at rest, due to changes in microcirculation. There was no change in the I/NI ratio during 201 Tl stress due to capillary recruitment, although HSA did demonstrate a decrease in the I/NI ratio with vasodilation, reflecting proximal vascular occlusion.

Using Laser Range-finding to Measure Bore Depth in Surgical Drilling of Bone.

Measuring drilled bore depth in bone is an important part of osteosynthesis surgery. Current methods have substantial limitations in terms of reliability, leading to placement of incorrectly sized screws and unsatisfactory user experience. (1) Can a prototype laser range-finder measure bore depth in bone as well as or better than a conventional depth gauge in terms of accuracy and precision (that is, variability)? A conventional analog orthopaedic surgical depth gauge was compared with a laser range-finder-based prototype. Experiments were conducted on four pig hind limbs, with bicortical holes drilled in the femur and the tibia. Two surgeons alternated drilling bores in three different clinically relevant conditions: straight drilling through the diaphysis, angled drilling through the diaphysis, and straight drilling through the metaphysis. Depth measurements were taken with the laser range-finder-based prototype, and the conventional depth gauge and compared against depth measurements obtained from a CT image that served as the reference measurement. In straight diaphyseal drilling the laser range-finder-based prototype had a larger mean error of 1.34 mm (± 0.7 mm) compared with a mean error of -0.06 mm (± 1.38 mm) using the conventional gauge (95% CI 0.824 to 1.976; p < 0.001). In angled diaphyseal drilling, there was no difference in mean error between the laser range-finder-based prototype (1.66 ± 0.86 mm) and the conventional gauge (2.36 ± 3.79 mm [95% CI -2.338 to 0.938]; p = 0.393). In straight metaphyseal drilling, there was no difference in mean error between the laser range-finder-based prototype (2.11 ± 0.8 mm) and the conventional gauge (1.51 ± 3.19 mm [95% CI -0.500 to 1.700]; p = 0.280). The laser range-finder-based prototype had greater precision (smaller variance) than the conventional depth gauge in straight diaphyseal drilling (p < 0.001), angled diaphyseal drilling (p < 0.001), and straight metaphyseal drilling (p < 0.001). A laser range-finder-based prototype mounted on a conventional surgical drill demonstrated overall similar accuracy and better precision in measuring drilled bore depth in bone compared with the conventional depth gauge. A device based on this concept could improve the reliability of bore depth measurement in surgical practice and could therefore reduce the frequency of screw replacement and associated complications.

Abstract 17160: Quantitative Characterization of Porcine Peripheral Artery Disease Models

Introduction: Peripheral artery disease (PAD) is a significant age-related medical condition affecting over 200 million patients worldwide. PAD cell therapy clinical trials have shown minimal benefits, and questions remain regarding the optimal cell type, dose, and delivery method. A large animal preclinical PAD model has not been standardized nor quantitatively characterized, potentially contributing to the lack of success in these trials. Hypothesis: We hypothesize that a durable model of PAD can be created in swine to test cell therapy efficacy as determined through quantitative measurements of microvascular muscle perfusion and limb functionality while walking. Methods: Swine were divided into three cohorts and underwent unilateral ligation of 1) the external iliac and circumflex iliac arteries, 2) the external, internal, and circumflex iliac arteries, or 3) distal hind limb arteries to create models of PAD. Automated gait analysis with a pressure-sensitive walkway was performed at various time points pre- and post-model creation to quantify functional deficits of trotting animals. Objective calculation of various gait parameters was performed by walkway software. A pulsed arterial spin labeling (ASL) MRI sequence was used to measure perfusion in pig hind limbs during cuff-induced reactive hyperemia. Fluoroscopy-based angiography was performed during model creation and sixth week terminal procedures, and fluorescent microsphere perfusion measurements were obtained for limb muscles at study conclusions. Results: The most severe, durable functional deficits were exhibited in Cohort 2. Cohort 1 exhibited transient functional abnormalities in ischemic vs. non-ischemic hind limbs that returned to baseline in one week. Animals in Cohort 3 also displayed slight deficits early that quickly resolved. ASL MRI successfully quantified microvascular perfusion in hind limb skeletal muscle. Extensive collateral formation in ischemic hind limbs was detected at the end of each study. Conclusions: External and internal iliac artery ligation is a promising strategy to establish a durable model of hind limb ischemia in which to test the efficacy of cell and other therapeutics, better facilitating the transition of PAD therapies from bench to bedside.

Local and distant trauma after hypervelocity ballistic impact to the pig hind limb.

The development of high-energy weapons could increase the velocity of projectiles to well over 1000 m/s. The nature of the injuries caused by the ballistic impact of projectiles at velocities much faster than 1000 m/s is unclear. This study characterizes the mechanical and biochemical alterations caused by high-speed ballistic impact generated by spherical steel ball to the hind limbs of the pig. That the local and distal injuries caused by hypervelocity ballistic impact to the living body are also identified. It is showed that the severity of the injury was positively correlated with the velocity of the projectile. And 4000 m/s seems to be the critical velocity for the 5.6 mm spherical steel ball, which would cause severe damage to either local or distal organs, as below that speed the projectile penetrated the body while above that speed it caused severe damage to the body. In addition, vaporization prevented the projectile from penetrating the body and the consequent pressure wave seems to be the causal factor for the distant damage.

Comparison of the Effect of Prostaglandin E(1), Prostacycline and Adenosine on Peripheral Vascular Resistance.

Prostacycline, prostaglandin E(1), and adenosine are highly effective vasodilators. These three drugs are widely used in the treatment of peripheral arterial occlusive disease. The aim of the study was to compare the vasodilatory potency of these substances in the isolated perfused guinea pig hind limb. After equilibration with Tyrode's solution and precontraction with noradrenaline 3µM, prostaglandin E(1) and adenosine were administered at dosages of 0.1, 0.3, and 1µM, whereas prostacycline was administered at a dosage of 0.01, 0.03 and 0.1µM. 0.01µM prostacycline, 0.1 µM prostaglandin E(1), and 0.1µM adenosine were the lowest dosages at which a significant vasodilation could be reached for each substance. The reduction of peripheral vascular resistance at comparable dosages of 0.1µM was 11.0 +/- 2.6% (x +/- SEM, n = 5) for adenosine, 12.0 +/- 1.0% (n = 5) for prostaglandin E(1), but 28.0 +/- 9.3% (n = 5) for prostacycline (p < 0.05 versus adenosine and prostaglandin E(1)). Even at a dosage of 0.01 µM prostacycline, a comparable reduction in peripheral vascular resistance (16.0 +/- 2.8%) could be reached, compared to a ten-fold higher dosage of prostaglandin E(1) and adenosine. At the highest concentration of 1 µM, the vasodilatory effect of adenosine was significantly less expressed, compared to that of prostaglandin E(1) (18.0 +/- 3.4% versus 33.0 +/- 4.7%). In summary, prostacycline, at a ten-fold lower concentration, showed comparable vasodilatory effects to adenosine and prostaglandin E(1). The rank order at the vasodilatory potency is prostacycline > prostaglandin E(1) > adenosine. </hea

Localization of the autonomic, somatic and sensory neurons innervating the cranial tibial muscle of the pig

The location of sympathetic, somatic and sensory neurons projecting to the cranial tibial muscle of the pig hindlimb was studied with the neuronal non-transynaptic tracer Fast Blue. Additionally, the number and the size of these neurons were determinated. The Fast blue, randomly applied to the cranial tibial muscle belly of 3 pigs, labelled sympathetic neurons in the ipsilateral L5-S3 and contralateral S1 sympathetic trunk ganglia and in the prevertebral caudal mesenteric ganglia of both sides. The somatic motoneurons were identified in the ipsilateral ventral horn of the S1 segment of spinal cord, while the sensory neurons were located in the ipsilateral L7-S1 spinal ganglia. The diameter of the multipolar sympathetic neurons oscillated between 26 and 46 microm in the sympathetic trunk ganglia and between 18 and 42 microm in the caudal mesenteric ganglia. The size of the multipolar spinal motoneurons oscillated between 33 and 102 microm. The size of the pseudounipolar sensory neurons oscillated between 23 and 67 microm. In all ganglia, the labelled neurons were localized at random and did not show a somatotopic distribution. Our results document a conspicuous autonomic innervation projecting to the "classic" skeletal cranial tibial muscle. Probably this innervation is destined to the muscle vessels.

Biomechanical study of four modified Kessler flexor tendon suture teclmiques

Objective To compare tensile strength of four different modified Kessler flexor tendon suture techniques. Methods Forty flexor digitorum profundus tendons of white pig hind limbs were transected and repaired using 4 suture techniques, with 10 tendons for each technique. The four suture techniques were Kessler, Double Kessler, Running suture and Kessler plus Running suture. After suturing, 2 mm gap formation force and failure load of the sutures were tested. One-way ANOVA was applied for statistic comparison of each technique. Results Double Kessler suture technique has the greatest tensile strength with its failure load, fonowed by Kessler plus Running suture, Kessler and Running suture. Kessler plus Running suture method had the greatest 2 mm gap formation force, followed by Double Kessler, Running suture and Kessler technique. Conclusion Kessler plus Running suture can better endure early finger active mobilization. Key words: Tendon injuries; Suture techniques; Biomechanics

A Preliminary Assessment of the Identification of Saw Marks on Burned Bone*

This study assesses the degree of modification to the saw mark characteristics of dismembered skeletal remains when exposed to a controlled outdoor fire of limited duration. The sample consists of 36 adult pig hind limbs which were dismembered fleshed. Six handsaws and six power saws were used, with three limbs dismembered and burned for each of the saw types. Results indicate that fire exposure affects the visibility and identifiability of saw mark striae. With the handsaws, the bow saw, hacksaw, and keyhole saw were consistently recognizable. In the power saw group, the saw marks of the jigsaw, reciprocating saw, and chainsaw remained identifiable. Although the bone ends exhibited thermal alterations, the false starts were well preserved with minimal damage. Given the parameters of this study, it is possible to identify the class of saw based on the diagnostic characteristics present on the cremated bones.

Prostaglandin E2 Inhibits Eosinophil Trafficking through E-Prostanoid 2 Receptors

The accumulation of eosinophils in lung tissue is a hallmark of asthma, and it is believed that eosinophils play a crucial pathogenic role in allergic inflammation. Prostaglandin (PG) E(2) exerts anti-inflammatory and bronchoprotective mechanisms in asthma, but the underlying mechanisms have remained unclear. In this study we show that PGE(2) potently inhibits the chemotaxis of purified human eosinophils toward eotaxin, PGD(2), and C5a. Activated monocytes similarly attenuated eosinophil migration, and this was reversed after pretreatment of the monocytes with a cyclooxygenase inhibitor. The selective E-prostanoid (EP) 2 receptor agonist butaprost mimicked the inhibitory effect of PGE(2) on eosinophil migration, whereas an EP2 antagonist completely prevented this effect. Butaprost, and also PGE(2), inhibited the C5a-induced degranulation of eosinophils. Moreover, selective kinase inhibitors revealed that the inhibitory effect of PGE(2) on eosinophil migration depended upon activation of PI3K and protein kinase C, but not cAMP. In animal models, the EP2 agonist butaprost inhibited the rapid mobilization of eosinophils from bone marrow of the in situ perfused guinea pig hind limb and prevented the allergen-induced bronchial accumulation of eosinophils in OVA-sensitized mice. Immunostaining showed that human eosinophils express EP2 receptors and that EP2 receptor expression in the murine lungs is prominent in airway epithelium and, after allergen challenge, in peribronchial infiltrating leukocytes. In summary, these data show that EP2 receptor agonists potently inhibit eosinophil trafficking and activation and might hence be a useful therapeutic option in eosinophilic diseases.

Bone weathering patterns of metatarsal v. femur and the postmortem interval in Southern Ontario

Twenty-five defleshed pig femora and 25 metatarsals were placed outdoors and observed over 291 days to establish: (1) bone weathering patterns for use in estimating time since death in Southern Ontario and (2) whether larger (femora) or smaller (metatarsals) bones provide a better indicator of time since death. Pig hind limbs were observed to determine a timeline for decomposition of soft tissues during the fall and winter. Ambient air temperature, humidity, precipitation, sunlight, soil pH, and freezing and thawing were considered as factors affecting the breakdown of bone. Weathering patterns were observed based on the extent of bleaching, amount of periosteum and soft tissues present, as well as the appearance of greasiness, cracking and flaking of cortical bone. Both entomological activity and climatic conditions affected soft tissue decomposition. Animal activity affected both the process of bone weathering and soft tissue decomposition, causing variability in sample decomposition and bone breakdown. The variation in microenvironment, partially caused by soil composition, introduced variability in bone weathering rates. Four bone weathering stages were established based on patterns observed. Femora proved to be more resilient and showed more degrees of change due to weathering, thus proving to be a better indicator of time since death than metatarsals.

A novel antagonist of CRTH2 blocks eosinophil release from bone marrow, chemotaxis and respiratory burst

Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) has been revealed to be a novel receptor for prostaglandin (PG) D(2), which is a major mast cell product released during the allergic response. The aim of this study was to analyze the effects of a newly developed small molecule antagonist of CRTH2, Cay10471, on eosinophil function with respect to recruitment, respiratory burst and degranulation. Chemotaxis of guinea pig bone marrow eosinophils and human peripheral blood eosinophils were determined using microBoyden chambers. Eosinophil release from bone marrow was investigated in the in situ perfused guinea pig hind limb preparation. Respiratory burst and degranulation were measured by flow cytometry. Cay10471 bound with high affinity to recombinant human and guinea pig CRTH2, but not DP, receptors. The antagonist prevented the PGD(2)-induced release of eosinophils from guinea pig bone marrow, and inhibited the chemotaxis of guinea pig bone marrow eosinophils and human peripheral blood eosinophils. Pretreatment with PGD(2) primed eosinophils for chemotaxis towards eotaxin, and this effect was prevented by Cay10471. In contrast, PGD(2) inhibited the C5a-induced up-regulation of CD63, a cellular marker of degranulation, in a Cay10471-sensitive manner. Finally, Cay10471 abolished the respiratory burst of eosinophils upon stimulation by PGD(2). These data further emphasize the importance of CRTH2 in eosinophil function and show that Cay10471 is a highly potent and selective antagonist of PGD(2)-induced eosinophil responses. Cay10471 might hence be a useful compound for the treatment of allergic diseases.

The Role of the Prostaglandin D2 Receptor, DP, in Eosinophil Trafficking

Prostaglandin (PG) D2 is a major mast cell product that acts via two receptors, the D-type prostanoid (DP) and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) receptors. Whereas CRTH2 mediates the chemotaxis of eosinophils, basophils, and Th2 lymphocytes, the role of DP has remained unclear. We report in this study that, in addition to CRTH2, the DP receptor plays an important role in eosinophil trafficking. First, we investigated the release of eosinophils from bone marrow using the in situ perfused guinea pig hind limb preparation. PGD2 induced the rapid release of eosinophils from bone marrow and this effect was inhibited by either the DP receptor antagonist BWA868c or the CRTH2 receptor antagonist ramatroban. In contrast, BWA868c did not inhibit the release of bone marrow eosinophils when this was induced by the CRTH2-selective agonist 13,14-dihydro-15-keto-PGD2. In additional experiments, we isolated bone marrow eosinophils from the femoral cavity and found that these cells migrated toward PGD2. We also observed that BWA868c inhibited this response to a similar extent as ramatroban. Finally, using immunohistochemistry we could demonstrate that eosinophils in human bone marrow specimens expressed DP and CRTH2 receptors at similar levels. Eosinophils isolated from human peripheral blood likewise expressed DP receptor protein but at lower levels than CRTH2. In agreement with this, the chemotaxis of human peripheral blood eosinophils was inhibited both by BWA868c and ramatroban. These findings suggest that DP receptors comediate with CRTH2 the mobilization of eosinophils from bone marrow and their chemotaxis, which might provide the rationale for DP antagonists in the treatment of allergic disease.

The effect of the K + agonist nicorandil on peripheral vascular resistance

Background The vasoactive effect of nicorandil on coronary arteries is well known. Nicorandil exerts its vasodilatory effect through a dual mechanism of action: involving on the one hand cyclic guanosine monophosphate (c GMP) as a nitrovasodilatator, and on the other hand, acting as a potassium channel opener. Objective To address the question if nicorandil works in peripheral arteries, its effect on peripheral vascular resistance was evaluated in isolated perfused guinea pig hind limbs. Methods A catheter was inserted via the distal aorta and common iliac artery. Perfusion pressure was monitored under constant perfusion with Tyrode's solution, therefore changes in perfusion pressure represent changes in vascular resistance. After stabilization precontraction of the peripheral vascular bed was achieved with noradrenaline 3 μM and nicorandil was added in concentrations of 1, 10 and 100 μM. The effect of nicorandil (1, 10 and 100 μM) was tested in the presence of l-NAME and glybenclamide. Results A significant reduction of vascular peripheral resistance was already achieved at a concentration of 1 μM nicorandil (30.3 ± 6.1%, mean S.E.M., p < 0.001). At a concentration of 100 μM nicorandil the reduction of peripheral vascular resistance was 94.4 ± 16.4%. Peripheral vascular resistance was less but nearly comparable reduced by nicorandil (100 μM) if the endothelial NO effect was inhibited by l-NAME (58.6 ± 18.6%) or if the ATP-dependent potassium channels were blocked by glybenclamide (56.4 ± 14.6%). Conclusions In peripheral arteries the nitrovasodilator effect of nicorandil is nearly comparable to the potassium agonistic effect, and the concentration, which is necessary to reduce peripheral vascular resistance significantly, is comparable with dosages necessary for reduction of coronary resistance.

Use of Tissue Ultrafiltration for Treatment of Compartment Syndrome

To demonstrate the efficacy of compartment syndrome ultrafiltration for the treatment of acute compartment syndrome in an animal model. Our hypothesis is the removal of interstitial fluid will result in a reduction of intramuscular pressure compared with untreated controls in a model of bilateral induced compartment syndrome. Controlled experimental model. Animal research facility. Three pairs of porcine hindlimbs. Acute compartment syndrome was created in the pig hindlimb by infusion of saline to maintain the intramuscular pressure 30 mm Hg greater than the animal's mean arterial pressure for 8 hours. After a 2-hour reperfusion interval, ultrafiltration (removal of fluid through 1 mm diameter porous catheters, connected to -500 mm Hg suction) was commenced in 1 limb only and continued for 9.5 hours. Intramuscular pressure, ultrafiltrate volume, ultrafiltrate and serum levels of creatine kinase and lactate dehydrogenase, histologic measurement of extracellular and intracellular edema, as well as the degree of cellular necrosis. Intramuscular pressure tended to be lower on the treated side at the end of the treatment period [treated leg: 9.3 +/- 4.0 mm Hg (+/- SE), control leg: 19.3 +/- 1.4 mm Hg, P = 0.03]. Analysis of ultrafiltrate fluid showed that levels of creatine kinase and lactate dehydrogenase were elevated compared with serum levels. Creatine kinase levels in serum were measured at 4150 +/- 780 U/L, whereas ultrafiltrate levels of creatine kinase were 28,700 +/- 17,700 U/L (+/- SE) (P = 0.1). Lactate dehydrogenase was measured at 1950 +/- 180 U/L in serum, but markedly elevated in ultrafiltrate [160,000 +/- 88,900 U/L (+/- SE), P = 0.05]. Quantification of cellular and interstitial dimensions showed no difference in control and experimental limbs. Quantification of the degree of muscle necrosis revealed 6.1 +/- 2.7% necrosis in the treated limb compared to 11.3 +/- 1.6% necrosis in the control group (P = 0.02, df = 2, 1-tailed paired t test). This pilot study demonstrates the feasibility of tissue ultrafiltration for reduction of intramuscular pressure in this porcine model. Further studies are underway. Compartment syndrome ultrafiltration may be useful prophylactically in patients at risk for acute compartment syndrome. Sampling of interstitial fluid and frequent measurement of intramuscular pressure may allow earlier diagnosis and treatment of acute compartment syndrome, whereas the reduction of tissue pressure by compartment syndrome ultrafiltration may prevent acute compartment syndrome from occurring. Additionally, compartment syndrome ultrafiltration will not hinder the ability of clinicians to use the clinical examination and pressure monitoring as the gold standard.

Optimization of regional intraarterial naked DNA-mediated transgene delivery to skeletal muscles in a large animal model

Effective gene therapy for muscular dystrophy will likely require intravascular administration. Although plasmid DNA (pDNA) contained within a large volume and rapidly infused into a major artery can achieve gene transfer within downstream muscles, this is associated with substantial muscle edema. Here we hypothesized that excessive edema-related increases in intramuscular pressure (IM pressure) developed during intraarterial pDNA injections could hinder successful gene delivery. Accordingly, we monitored IM pressure during injection of pDNA carrying a LacZ transgene into the femoral artery of rats and pigs. Large variations in IM pressure were found between different muscles. There was a significant inverse relationship between IM pressure and the subsequent level of gene transfer to muscle. Modification of the injection protocol to reduce IM pressure led to greatly increased pDNA-mediated gene expression and reduced muscle damage in pigs. Under the most optimized conditions, average transfection within eight different muscles of the pig hind limb amounted to 22% of all fibers, attaining a maximum of 60% in the gastrocnemius muscle. We conclude that IM pressure monitoring is a simple and useful procedure, which can be applied in both small and large animals to help optimize pDNA-mediated gene transfer to skeletal muscles by the intraarterial route.

Nonviral monocyte chemoattractant protein-1 gene transfer improves arteriogenesis after femoral artery occlusion.

Local infusion of recombinant monocyte chemoattractant protein-1 (MCP-1) has been shown to enhance collateral artery formation in rabbit and pig hindlimb models. Owing to clinical disadvantages of protein infusion, a nonviral, liposome-based MCP-1 gene transfer was developed. Collateralization in a porcine hindlimb model served to provide a proof-of-principle for the functional benefit of MCP-1 overexpression. Development of arterial conductance as a measure of functionally relevant collateralization was evaluated in occluded as well as untreated hindlimbs in each animal. At the time of occlusion, MCP-1 and control DNA/DC-30 lipoplexes were transferred to femoral arteries of Goettingen minipigs (two therapeutic MCP-1 groups: 2 and 4 microg and one control group), using the Infiltrator local drug-delivery device. At 2 weeks following occlusion, collateralization was determined as changes in peripheral haemodynamic conductance, peripheral over aortic blood pressure ratio and angiographically visible morphology of the peripheral vessel tree. Nonviral MCP-1 gene transfer significantly improved peripheral conductance (control 11.69+/-2.78%, 2 microg 23.81+/-2.81%, P<0.05 and 4 microg 23.36+/-3.1%, P<0.05; n=12 per group) as well as the ratio of peripheral over aortic blood pressure (control 0.64+/-0.03%, 2 microg 0.75+/-0.02%, P<0.05 and 4 mug 0.75+/-0.02%, P<0.05; n=12 per group) when compared to the untreated controls 2 weeks after occlusion. Thus, it could be demonstrated for the first time that in situ overexpression of MCP-1 following local nonviral gene transfer is a potential approach to improve peripheral collateralization.

Graded alterations of RBC aggregation influence in vivo blood flow resistance.

Although the effects of red blood cell (RBC) aggregation on low-shear rate blood viscosity are well known, the effects on in vivo flow resistance are still not fully resolved. The present study was designed to explore the in vivo effects of RBC aggregation on flow resistance using a novel technique to enhance aggregation: cells are covalently coated with a block copolymer (Pluronic F-98) and then suspended in unaltered plasma. RBC aggregation was increased in graded steps by varying the Pluronic concentration during cell coating and was verified by microscopy and erythrocyte sedimentation rate (ESR), which increased by 200% at the highest Pluronic level. RBC suspensions were perfused through an isolated in situ guinea pig hindlimb preparation while the arterial perfusion pressure was held constant at 100 mmHg via a pressure servo-controlled pump. No significant effects of enhanced RBC aggregation were observed when studies were conducted in preparations with intact vascular control mechanisms. However, after inhibition of smooth muscle tone (using 10(-4) M papaverin), a significant change in flow resistance was observed in a RBC suspension with a 97% increase of ESR. Additional enhancements of RBC aggregation (i.e., 136 and 162% increases of ESR) decreased flow resistance almost to control values. This was followed by another significant increase in flow resistance during perfusion with RBC suspensions with a 200% increase of ESR. This triphasic effect of graded increases of RBC aggregation is most likely explained by an interplay of several hemodynamic mechanisms that are triggered by enhanced RBC aggregation.

Elevated fluid shear stress enhances postocclusive collateral artery growth and gene expression in the pig hind limb.

The role of fluid shear stress (FSS) in collateral vessel growth remains disputed and prospective in vivo experiments to test its morphogenic power are rare. Therefore, we studied the influence of FSS on arteriogenesis in a new model with extremely high levels of collateral flow and FSS in pig and rabbit hind limbs. A side-to-side anastomosis was created between the distal stump of one of the bilaterally occluded femoral arteries with the accompanying vein. This clamps the collateral reentry pressure at venous levels and increases collateral flow, which is directed to a large part into the venous system. This decreases circumferential wall stress and markedly increases FSS. One week after anastomosis, angiographic number and size of collaterals were significantly increased. Maximal collateral flow exceeded by 2.3-fold that obtained in the ligature-only hind limb. Capillary density increased in lower leg muscles. Immunohistochemistry revealed augmented proliferative activity of endothelial and smooth muscle cells. Intercellular adhesion molecule-1 and vascular cell adhesion molecule (VCAM)-1 were upregulated, and monocyte invasion was markedly increased. In 2-dimensional gels, actin-regulating cofilin1 and cofilin2, destrin, and transgelin2 showed the highest degree of differential regulation. High levels of FSS cause a strong arteriogenic response, reinstate cellular proliferation, stimulate cytoskeletal rearrangement, and normalize maximal conductance. FSS is the initiating molding force in arteriogenesis. The role of fluid shear stress on the development of a collateral circulation was studied by abruptly increasing collateral blood flow by a distal femoral artery-to-vein anastomosis. This increased number and size of collateral vessels to a hitherto unknown degree. Fluid shear stress is the primary and strongest arteriogenic stimulus.

The effect of caffeine on peripheral vascular resistance in isolated perfused guinea pig hind limbs.

The role of caffeine in cardiovascular disease is controversial. Most of its pharmacologic actions are attributed to its role as an adenosine antagonist. Adenosine is one of the most important endogenous vasodilatative substances and is released under ischemic conditions, for example, in the skeletal muscle of patients with peripheral arterial occlusive disease. We aimed to investigate the influence of caffeine on peripheral vascular resistance and on the beneficial vasodilatory effect of adenosine in isolated perfused guinea pig hind limbs. (1) Caffeine was administered at 0.5, 5, and 50 micromol/L under normoxic conditions. (2) The vasculature of the perfused guinea pig hind limb was precontracted with noradrenaline (3 micromol/L), followed by adenosine (10 micromol/L) under normoxic conditions. When vascular resistance (VR) had reached a steady state, caffeine was administered additionally at dosages of 0.5, 5, and 50 micromol/L. (3) This protocol was repeated using iloprost 0.1 micromol/L instead of adenosine as vasodilatory substance. (4) Under hypoxia, caffeine was again administered at the above dosages. (5) Under hypoxia, experiments with adenosine A2-receptor antagonists (alloxazine 10 micromol/L and ZM 241385 100 nmol/L) were done. Under normoxic conditions, 0.5 and 5 micromol/L caffeine had nearly no effect on vascular resistance compared with baseline conditions. A slight, but statistically not significant decrease in VR was achieved with 50 micromol/L caffeine. In the presence of noradrenaline, the vasodilatory effect of adenosine was reduced by 7.6 +/- 1.6% after the addition of 0.5 micromol/L caffeine, and by 37.3 +/- 3.8% at a dosage of 5 micromol/L caffeine. A dosage of 50 micromol/L caffeine completely abolished the vasodilatative effect of adenosine. In the presence of iloprost, only a slight but statistically insignificant inhibitory influence (0.9%) of caffeine at a dosage of 50 micromol/L could be seen. Hypoxia significantly reduced VR. Caffeine at 0.5 micromol/L diminished this effect by about 53.2 +/- 4.6% and abolished it at 5 and 50 micromol/L. The hypoxia-induced adenosine-mediated vasodilatation seems to be an adenosine A2A-receptor-mediated effect. The observed effect of hypoxia-induced vasodilatation in peripheral arteries may be the result of the vasodilatory effect of elevated endogenous adenosine during hypoxia. For patients with peripheral arterial disease, drinking of caffeine-containing beverages may reduce the beneficial vasodilatory effect of elevated endogenous adenosine levels.