Validity of fluorescent microspheres method for bone blood flow measurement during intentional arterial hypotension.

In this study, we compared bone blood flow values obtained by simultaneously injected fluorescent (FM) and radiolabeled microspheres (RM) at stepwise reduced arterial blood pressure. Ten anesthetized female New Zealand White rabbits received simultaneous left ventricular injections of FM and RM at 90, 70, and 50 mmHg mean arterial blood pressure (MAP). After the experiments, both kidneys and long bones of all four limbs were removed and dissected in a standardized manner. Radioactivity (corrected for decay, background, and spillover) and fluorescence were determined, and blood flow values were calculated. Relative blood flow values estimated for each bone sample by RM and FM were significantly correlated (r = 0.98, slope = 0.99, and intercept = 0.04 for 90 mmHg; r = 0.98, slope = 0.94, and intercept = 0.09 for 70 mmHg; r = 0.98, slope = 0.96, and intercept = 0.07 for 50 mmHg). Blood flow values (ml x min-1 x 100 g-1) of right and left bone samples determined at the different arterial blood pressures were identical. During moderate hypotension (70 mmHg MAP), blood flow in all bone samples remained unchanged compared with 90 mmHg MAP, whereas a significant decrease of bone blood flow was observed at severe hypotension (50 mmHg MAP). Our results demonstrate that the FM technique is valid for measuring bone blood flow. Differences in bone blood flow during altered hemodynamic conditions can be detected reliably. In addition, changes in bone blood flow during hypotension indicate that vasomotor control mechanisms, as well as cardiac output, play a role in setting bone blood flow.     

Physiological and mechanical adaptation of periarticular cancellous bone after joint ligament injury.

The relation between blood flow and bone mineral density (BMD) of periarticular bone was examined in an in vivo model of joint instability. Eighty mature New Zealand White rabbits were randomly assigned to experimental [anterior cruciate ligament transection (ACLX)], sham-operated control, or age-matched normal control groups. Experimental rabbits underwent unilateral transection of the right anterior cruciate ligament, and the nonoperated left [contralateral (Cntra)] limb was a within-animal control. BMD and blood flow to the periarticular bone in the femoral condyles were assessed in each group at 2, 4, 6, 14, and 48 wk postsurgery, using quantitative computed tomography scanning and entrapment of colored microspheres. BMD was significantly lower (5%) in the ACLX compared with Cntra limbs. Periarticular bone blood flow in the ACLX limbs was significantly greater than in the Cntra limb (29%) in the early stages (6 wk) after injury. Up to 48 wk post-ACLX, a significant correlation was found between increased blood flow and decreased BMD in the periarticular bone of the femoral condyles in the ACLX limbs. This correlation suggested that heightened blood flow may be linked to mechanisms of bone adaptation in joints after ligament injury.     

The evaluation of knee bone mineral density following open-wedge high tibial osteotomy

Examinations of bone density changes in selected knee bone ends were evaluated prospectively in a randomized group of 28 patients, aged from 41 to 65 (mean: 55.3 years), who had varus deformations of their mechanic limb axes, mean 8 degrees. The examinations were conducted during the preoperative period, 10 days, 3, 6, and 12 weeks, as well as 6 and 12 months after the procedure. A statistically significant increase in bone density was observed in the medial tibial condyle area, while a statistically insignificant decrease of bone density was noted in the medial femoral condyles. Bone density increased in the lateral tibial condyle area, whereas there were no density changes in the area of the lateral femoral condyles. The research results demonstrate that the relief achieved in ailments after high tibial osteotomies does not directly correspond to the bone density of the affected areas.     

Pathways of load-induced cartilage damage causing cartilage degeneration in the knee after meniscectomy

Results of both clinical and animal studies show that meniscectomy often leads to osteoarthritic degenerative changes in articular cartilage. It is generally assumed that this process of cartilage degeneration is due to changes in mechanical loading after meniscectomy. It is, however, not known why and where this cartilage degeneration starts. Load induced cartilage damage is characterized as either type (1)--damage without disruption of the underlying bone or calcified cartilage layer--or type (2), subchondral fracture with or without damage to the overlying cartilage. We asked the question whether cartilage degeneration after meniscectomy is likely to be initiated by type (1) and/or type (2) cartilage damage. To investigate that we applied an axisymmetric biphasic finite element analysis model of the knee joint. In this model the articular cartilage layers of the tibial and the femoral condyles, the meniscus and the bone underlying the articular cartilage of the tibia plateau were included. The model was validated with data from clinical studies, in which the effects of meniscectomy on contact areas and pressures were measured. It was found that both the maximal values and the distributions of the shear stress in the articular cartilage changed after meniscectomy, and that these changes could lead to both type (1) and type (2) cartilage damage. Hence it likely that the cartilage degeneration seen after meniscectomy is initiated by both type (1) and type (2) cartilage damage.     

High-frequency, low-magnitude vibration does not prevent bone loss resulting from muscle disuse in mice following botulinum toxin injection

High-frequency, low-magnitude vibration enhances bone formation ostensibly by mimicking normal postural muscle activity. We tested this hypothesis by examining whether daily exposure to low-magnitude vibration (VIB) would maintain bone in a muscle disuse model with botulinum toxin type A (BTX). Female 16-18 wk old BALB/c mice (N = 36) were assigned to BTX-VIB, BTX-SHAM, VIB, or SHAM. BTX mice were injected with BTX (20 μL; 1 U/100 g body mass) into the left hindlimb posterior musculature. All mice were anaesthetized for 20 min/d, 5 d/wk, for 3 wk, and the left leg mounted to a holder. Through the holder, VIB mice received 45 Hz, ± 0.6 g sinusoidal acceleration without weight bearing. SHAM mice received no vibration. At baseline and 3 wk, muscle cross-sectional area (MCSA) and tibial bone properties (epiphysis, metaphysis and diaphysis) were assessed by in vivo micro-CT. Bone volume fraction in the metaphysis decreased 12 ± 9% and 7 ± 6% in BTX-VIB and BTX-SHAM, but increased in the VIB and SHAM. There were no differences in dynamic histomorphometry outcomes between BTX-VIB and BTX nor between VIB and SHAM. Thus, vibration did not prevent bone loss induced by a rapid decline in muscle activity nor produce an anabolic effect in normal mice. The daily loading duration was shorter than would be expected from postural muscle activity, and may have been insufficient to prevent bone loss. Based on the approach used in this study, vibration does not prevent bone loss in the absence of muscle activity induced by BTX.     

Different effects of oestradiol benzoate and norethisterone on the blood flow and mineral content in rat bones.

In three experiments (2 on females, 1 on males), we determined the blood flow in the tibia and the distal part of the femur, together with cardiac output (by means of 85Sr-microspheres), tibial bone density and tibial ash weight related to bone volume. We found that 1) the bone blood flow always fell significantly after oestradiol benzoate, 2) no change occurred after norethisterone in doses corresponding to those of oestradiol benzoate, but the blood flow showed a tendency to fall after doses one order higher (it decreased significantly in one case only), 3) the density of the tibia and tibial ash weight related to bone volume rose nonsignificantly after oestradiol benzoate, but fell (mostly statistically significantly) after norethisterone. The lowering of the bone mineral indexes in rat bones after norethisterone is a surprising and potentially significant finding requiring further verification.     

Periarticular cancellous bone changes following anterior cruciate ligament injury.

To understand more fully the early bone changes in an experimental model of osteoarthrosis, we quantified periarticular bone mineral density and bone mechanical properties in anterior cruciate ligament transected (ACLX) knee joints (4, 10, 32, and 39 wk post-ACLX) compared with contralateral joints and unoperated normal joints of skeletally mature animals. Maximal stress and energy were significantly reduced in ACLX cancellous bone from the medial femoral condyles at 4 wk postinjury. All mechanical properties (e.g., yield stress and elastic modulus) declined after 4 wk and were significantly reduced at 10 wk. ACLX bone mineral density was significantly reduced at all measured time points. Ash content was significantly reduced at 10 and 32 wk. Changes in the lateral condyles were similar but less pronounced than in the medial condyles. These bony changes accompanied the earliest articular cartilage molecular changes and preceded changes in the articular cartilage gross morphology. We suggest that these early changes in bone mechanical behavior contribute to the progression of osteoarthrosis and pathogenic changes in the joint.     

Arterial hypoxia does not affect subchondral pressure and regional blood flow

The influence of arterial hypoxia on bone marrow pressure, regional blood flow and oxygen and carbon dioxide tensions was investigated by simultaneous and continuous measurements in the femoral condyles of 8 rabbits. Arterial hypoxia was obtained by hypoventilation. The subchondral gas tensions and regional blood flow were measured by a previously described technique based on mass spectrometry. Arterial hypoxia caused a significant decrease in subchondral oxygen tension and an increase in subchondral carbon dioxide tension. There was no significant change in bone marrow pressure and regional blood flow.     

The role of the cardiac-pulmonary blood volume in the changes in left ventricle output during stimulation of the afferent fibers of somatic nerves

Stimulation of tibial nerve afferent fibers has revealed heterogeneous shifts of left ventricular output, as well as pulmonary artery and posterior vena cava blood flow in anesthetized cats. Uniform changes in left ventricular output and pulmonary artery blood flow were noted in the majority of cases, with venous return most often exceeding pulmonary artery blood flow. beta-adrenoreceptor blockade failed to influence changes in pulmonary artery blood flow. It is concluded that the increase in pulmonary artery blood flow depends on the rise in venous return, but not on neurogenic influence upon the right ventricle. The reduction in left ventricular output is the result of decreased right ventricular outflow due to its overload caused by pulmonary vasoconstriction.     

The effect of toxic doses of cholecalciferol (Vitamin D3) on the serum zinc levels in rats

Zinc is a trace element important to bone mineralization as well as, in general, nutrition. It is known that cholecalciferol (vitamin D₃) affects bone metabolism. In this study toxic doses of vitamin D₃ were injected subcutaneously (25 μg/d) to rats for 5 wk. It caused a significant increase in serum zinc levels (p<0.02). On the other hand, no significant increase was detected in the other groups. Excessive amounts of vitamin D₃ caused bone breakdown and increased the levels of zinc in blood.     

Direct assessment and distribution of regional portal blood flow in the pig by means of fluorescent microspheres.

Measurement of regional organ blood flow by means of fluorescent microspheres (FM) is an accepted method. However, determination of regional portal blood flow (RPBF) cannot be performed by microspheres owing to the entrapment of the spheres in the upstream capillary bed of the splanchnic organs. We hypothesized that an adequate experimental setting would enable us to measure RPBF by means of FM and to analyze its distribution within the pig liver. A mixing chamber for the injection of FM was developed, and its capability to distribute FM homogeneously in the blood was evaluated in vitro. The chamber was implanted into the portal vein of six anesthetized pigs (23.5 +/- 2.9 kg body wt). Three consecutive, simultaneous injections of FM of two different colors into the chamber were performed. Reference portal blood samples were collected by means of a Harvard pump. At the end of the experiment, the liver was explanted and fixed in formalin before dissection. FM were isolated from the tissue samples by an automated process, and fluorescence intensity was determined. Comparison of 5,458 single RPBF values, determined by simultaneously injected FM, revealed good agreement (bias 2.5%, precision 12.7%) and high correlation (r = 0.97, r2 = 0,95, slope = 1.04, intercept = 0.05). Median RPBF was 1.07 +/- 0.78 ml x min(-1) x g(-1). Allocation of the blood flow values to the anatomic regions of the liver revealed a significantly higher RPBF (P = 0.01) in the liver tissue located close to the diaphragm compared with the rest of the organ and a significantly lower RPBF (P = 0.01) in the left liver lobe compared with the median and right lobes. The results show that the model presented makes it possible to measure RPBF by means of FM reliably and that RPBF is distributed heterogeneously in the porcine liver.     

Depletion of eosinophils by anti-IL-5 monoclonal antibody treatment of mice infected with Trichinella spiralis does not alter parasite burden or immunologic resistance to reinfection.

Mechanisms of parasite killing by eosinophils are widely studied and are often implicated in mediating resistance to parasitic infection, especially in conjunction with specific antibodies. Evidence for the eosinophil as an anti-parasite killer cell in vivo is limited and may not justify the belief that eosinophils engage and/or kill infective helminths. We reexamined this question in a mouse model of trichinosis in which antisera to eosinophils were previously used to show the requirement for eosinophils in resistance to this nematode. The current studies used mAb to IL-5 to suppress eosinophil levels in CF1 mice infected with Trichinella spiralis. In mice given a primary infection and injected with an isotype control mAb or left untreated, the medullary and peripheral blood eosinophil numbers peaked at 3 wk postinfection (PI) and returned to baseline levels by 4 wk PI. Peripheral blood eosinophil numbers in infected mice injected with anti-IL-5 were maintained at levels below those of uninfected normal mice through 4 wk of infection. Histologically, there was a prominent eosinophil accumulation in infected, untreated, or control-mAb-treated mice associated with nurse cell complexes containing infective juveniles in skeletal muscle at 3 and 4 wk PI. This was largely eliminated in mice treated with anti-IL-5 mAb. However, the number of muscle stage juvenile worms recovered 3 and 4 wk PI after acid pepsin digestion was unaffected by eosinophil depletion. Challenge infections, in which mice were infected at day 0 with 125 muscle stage worms and challenged at day 28 PI with 350 muscle stage worms, developed peak eosinophil numbers in bone marrow and peripheral blood 3 wk after primary infection and 2 wk after challenge infection in mice receiving either no treatment or control mAb. In challenged mice receiving anti-IL-5 mAb, medullary and peripheral blood eosinophil numbers remained at or below those of uninfected animals. Although all groups exhibited significant resistance measured as muscle stage worm burdens 56 days PI, eosinophil depletion did not affect resistance of muscle worm recovery. These results suggest that eosinophils are not essential in the control of T. spiralis in either primary or challenge infections of CF1 mice. This in vivo study illustrates the questionable value of in vitro killing assays to assign effector function to any single inflammatory cell type.     

Changes in mineralization and biomechanics of tibial metaphyses in splinted rats.

The effect of 3 wk of splintage of a single hindlimb on the midarea and mineral content of both tibial metaphyses was assessed immediately after splint removal and after 1 mo of mobilization in 12-wk-old Sprague-Dawley rats. Immobilization reduced tibial metaphyseal bone mineral density (BMD) in immobilized limbs compared with "free" limbs of splinted animals and with controls. These changes persisted and were accentuated by relatively greater increases in tibial metaphyseal BMDs of unsplinted (control) animals after 7 wk. Immediately after splintage, tibial metaphyseal areas and total mineral contents of both hindlimbs of splinted animals were reduced compared with those of unsplinted animals. However, the relationship between mineralization and area differed between the free and immobilized limbs of splinted animals. The breaking strain and the breaking energy of immobilized and free femurs of splinted animals were impaired 4 wk after the removal of the splint. This impairment was correlated with an effect of splintage on femoral size with some additional local effect from immobilization. Thus osteoporotic changes consequent on immobilization include both local effects on mineralization and general effects on growth, which may separately influence the elastic properties of bone.     

Effects of eccentric exercise training on cortical bone and muscle strength in the estrogen-deficient mouse.

The purpose of this study was to determine whether eccentrically biased exercise training could attenuate changes in muscle and bone function associated with estrogen deficiency in the mouse model. Four groups of ICR mice were used: control (Con), sham ovariectomized (Sham), ovariectomized (OVX), and ovariectomized + high-force resistance training (OVX+Train). All groups except Con were implanted with a nerve cuff surrounding the peroneal nerve to stimulate the left ankle dorsiflexors. Training consisted of 30 stimulated eccentric contractions of the left ankle dorsiflexors at approximately 150% of peak isometric torque every third day for 8 wk. After the training period, groups were not significantly different with regard to peak torque or muscle size. However, the tibial midshaft of the trained leg in the OVX+Train mice exhibited greater stiffness (+15%) than that in the untrained OVX mice, which could not be explained by changes in cross-sectional geometry of the tibia. Scaling of bone mechanical properties to muscle strength were not altered by ovariectomy or training. These data indicate that eccentric exercise training in adult mice can significantly increase bone stiffness, despite the absence of ovarian hormones.     

Chondrocyte apoptosis enhanced at the growth plate: a physeal response to a diaphyseal fracture

Post-traumatic overgrowth of growing long bones is a common clinical phenomenon in paediatric traumatology and is the result of an enhanced stimulation of the nearby growth plate after fracture. To date, the exact post-fractural reactions of the growth plate are poorly understood. The aim of this study has been to determine the impact of fracture on the frequency of chondrocyte apoptosis of the growth plate. Rats sustained a mid-diaphyseal closed fracture of the left tibia or were left untreated. All animals were killed 3, 10, 14 or 29 days after trauma. The left and right tibiae were harvested and apoptotic chondrocytes of the proximal tibial growth plate were detected by TUNEL staining. The apoptosis percentage of physeal chondrocytes was statistically compared among fractured bones, intact contra-lateral bones and control bones. The physeal apoptosis rate of the fractured bone was significantly higher than that of the contra-lateral intact bone (valid for all evaluated days) and the control bone (valid from day 10 onwards). Contra-lateral intact tibiae never showed significantly higher apoptosis rates compared with control tibiae. Thus, mid-diaphyseal fracture influences the nearby growth plate by stimulating chondrocyte programmed cell death, which is associated with cartilage resorption and bone replacement. The lack of a significant difference between the intact contra-lateral and the intact control bone suggests that fracture only has a local effect that contributes to the greater apoptosis rate of the adjacent physis.     

Evaluation of laser-Doppler perfusion imaging for measurement of blood flow in cortical bone.

Most techniques currently available to measure blood flow in bone are time consuming and require destruction of the tissue, but laser-Doppler technology offers a less invasive method. This study assessed the utility of laser-Doppler perfusion imaging (LDI) to measure perfusion in cortical bone. Twelve mature New Zealand White rabbits were assigned to one of three groups: normal control, constriction (norepinephrine), or dilatation (nitroprusside). The left and right medial tibiae were consecutively scanned at red (634-nm) and near-infrared (810-nm) wavelengths to examine the repeatability of LDI output. The pharmacological intervention groups were injected with the respective drug, and LDI measurements at 810 nm were obtained concurrently with colored microsphere-determined flow in all of the groups. LDI effectively quantified blood flow in cortical bone and detected physiologically induced changes in perfusion. A significant positive correlation was found between microsphere-determined flow and LDI output (r = 0.6, P < 0.05). Repeatability of consecutive LDI measurements was within 5%. The effectiveness of LDI to measure perfusion in bone suggests this method has potential for investigating the role of blood flow in bone metabolism and remodeling.     

Cryptosporidial infections in SCID mice reconstituted with human or murine lymphocytes.

Severe combined immunodeficient (SCID) mice were experimentally infected with Cryptosporidium parvum. Adoptive transfer of BALB/c thymocytes, spleen and bone marrow cells resulted in functional immunologic reconstitution followed by complete eradication of the cryptosporidial infection. Additional SCID mice were injected with human blood peripheral blood lymphocytes and were subsequently infected with C. parvum. The latter mice (SCID-hu-PBL) were at least partially reconstituted with human lymphoid tissues, as evidenced by flow cytometric identification of human cell populations in the SCID mouse spleens and the response of these cells to the T-cell mitogen phytohemagglutinin. The SCID-hu-PBL mice did not resolve the cryptosporidial infections, although a transient reduction in parasitemia was noted 4-6 wk post-reconstitution.     

The free thoracoumbilical flap for resurfacing large soft-tissue defects of the lower extremity

Both cadavers and living patients were studied regarding a method to resolve large skin defects with bone exposure in the leg, with long-distance thrombosis of the anterior tibial vessels or posterior tibial vessels resulting from traumatic lesions. Forty-six casting mold specimens of cadaveric legs were investigated. There were rich communication branches among the anterior tibial artery, posterior tibial artery, and fibular artery in the foot and ankle, which complemented each other well. Twenty-six patients with large skin defects with bone exposure in the proximal or middle segment of the leg were admitted to the authors' hospital. Among those patients, 19 demonstrated long-distance thrombosis of the anterior tibial vessels or posterior tibial vessels resulting from traumatic lesions. During treatment, a thoracoumbilical flap based on the inferior epigastric vessels was anastomosed to the distal stump of the anterior tibial vessels or the posterior tibial vessels, with reversed flow. All defects were successfully repaired, with good color and texture matches of the flaps. This method can be used for patients with normal anterior tibial vessels or posterior tibial vessels, normal distal stumps of the injured blood vessels, and good reversed flow. The method has the advantages of dissecting blood vessels in the recipient area during the débridement, not affecting the blood circulation of the injured leg, not sacrificing blood vessels of the opposite leg, and not fixing the patient in a forced posture. The muscles are less bulky in the distal one-third of the leg, and the blood vessels are shallow and can be dissected and anastomosed easily. When the flap is used for reconstruction in the proximal two-thirds of the leg, the blood vessel pedicle of the free flap is at a straight angle, without kinking.     

Gene therapy for chronic myocardial ischemia using platelet-derived endothelial cell growth factor in dogs

Platelet-derived endothelial cell growth factor (PD-ECGF), also known as thymidine phosphorylase (TP), has been reported to possess angiogenic activity and to inhibit apoptosis. This study was performed to determine whether PD-ECGF/TP can be used to ameliorate chronic myocardial ischemia. Myocardial ischemia was created in 40 mongrel dogs by placement of an ameroid constrictor on the proximal left anterior descending coronary artery (LAD). Plasmid vector encoding human PD-ECGF/TP cDNA (pCIhTP group; n = 12), empty vector pCI (pCI group; n = 12), or saline (Saline group; n = 12) was directly injected into the LAD territory 3 wk after ameroid constrictor implantation. Myocardial blood flow was detected using PET at baseline, 3 wk after ameroid constrictor implantation, and 2 wk after therapeutic treatment. At the end of the experiment, the hearts were isolated for biological and histological analysis. In the pCIhTP group, the transfected heart strongly expressed PD-ECGF/TP. The size of the infarct was smaller in the pCIhTP group than in the pCI or Saline group. The number of apoptotic myocardial cells was decreased in the pCIhTP group compared with the control groups based on triple immunohistochemical staining for von Willebrand factor, alpha-actin smooth muscle cells, and single-strand DNA. The level of proapoptotic protein Bax markedly decreased in the pCIhTP group compared with the other groups. Double immunohistochemical staining for von Willebrand factor and alpha-actin smooth muscle cells demonstrated that angiogenesis and arteriogenesis occurred, and paralleled the changes in myocardial blood flow and myocardial function in the pCIhTP group. We conclude that genetic approaches using PD-ECGF/TP to target the myocardium are effective for alleviating chronic myocardial ischemia.     

Blood flow in different regions of bone marrow after short-term exercise

Total and regional blood flow was studied in femoral bone marrow of rats exposed to a short-term exhaustive exercise. The 10 micron microspheres labeled with 58Co were injected into femoral artery and the subsequent radioactivity yield was measured in 3 regions of the bone marrow (proximal metaphysis, diaphysis and distal metaphysis). Following exercise a rise of blood flow in the metaphyseal regions was accompanied by a fall in the diaphyseal area. The changes did not alter, however, the total blood flow rate in bone marrow which remained constant irrespective of the heavy exercise load.