Theoretical principles underlying optical stimulation of myelinated axons expressing channelrhodopsin-2.

Numerous clinical conditions can be treated by neuromodulation of the peripheral nervous system (PNS). Typical electrical PNS therapies activate large diameter axons at lower electrical stimulus thresholds than small diameter axons. However, recent animal experiments with peripheral optogenetic neural stimulation (PONS) of myelinated axons expressing channelrhodopsin-2 (ChR2) have shown that this technique activates small diameter axons at lower irradiances than large diameter axons. We hypothesized that the small-to-large diameter recruitment order primarily arises from the internodal spacing relationship of myelinated axons. Small diameter axons have shorter distances between their nodes of Ranvier, which increases the number of nodes of Ranvier directly illuminated relative to larger diameter axons. We constructed "light-axon" PONS models that included multi-compartment, double cable, myelinated axon models embedded with ChR2 membrane dynamics, coupled with a model of blue light dynamics in the tissue medium from a range of different light sources. The light-axon models enabled direct calculation of threshold irradiance for different diameter axons. Our simulations demonstrate that illumination of multiple nodal sections reduces the threshold irradiance and enhances the small-to-large diameter recruitment order. In addition to addressing biophysical questions, our light-axon model system could also be useful in guiding the engineering design of optical stimulation technology that could maximize the efficiency and selectivity of PONS.

Effects of immobilization on the biomechanical properties of the broiler tibia and gastrocnemius tendon.

Researchers have provided much insight into the various factors that influence the incidence of musculoskeletal problems in the poultry industry. However, a better understanding of the mechanobiology of broiler bone and tendon can have a positive effect on the welfare of the production bird and assist in the development of improved production practices. This study investigated the mechanical adaptability responses due to disuse on the biomechanical properties of the broiler tibia and gastrocnemius tendon. Beginning at 3 wk of age, broilers were placed in a harness system designed to eliminate load bearing of the leg. After 2 wk of this treatment, the average values for body mass and shank length of the birds were 58 and 85% of the values for the controls, respectively. The treatment reduced the mineral content of the tibia by approximately 50%, tibia structural strength by 40%, and tibia material strength by 8%. The structural strength and toughness of the gastrocnemius tendon were reduced by 10 and 30%, respectively, whereas the material strength, material toughness, and material stiffness of the tendon increased by approximately 75, 65, and 70%, respectively.

Effects of activity on avian gastrocnemius tendon.

Physical activity and its relationship to animal health is a continuous concern of the food animal industry. This study investigated the relationship between broiler (meat-type chicken) activity to the structural integrity of the gastrocnemius tendon. Birds were exposed to treadmill pacing to determine if increased mobilization would increase tendon strength and improve its resistance to soft tissue injury. One hundred eighty broilers raised under normal commercial housing conditions were forced to walk on a treadmill 30 min/d, 5 d/wk for 3 wk, beginning at 3 wk of age. The treadmill treatment did affect the growth rate of the broilers. At the end of the study, the average body mass of the treatment birds was 9% less than the average body mass of the control birds, and the average length of the treatment shanks was 5% less than those from the control birds. Biomechanical parameters were measured and used to determine changes in the structural and material integrity of the tendons. The treadmill treatment did not affect tendon toughness, stiffness, relaxation behavior, and failure strength, but treatment did appear to affect tendon geometry, in which 33% of the treadmill treatment tendons had an increased amount of tissue near the bifurcation. The treadmill treatment did not affect the amount of procollagen within the tendon, and no cellular anomalies were found.

In vitro culture decreases the expression of TGF(beta), Hsp47 and type I procollagen and increases the expression of CTGF in avian tendon explants.

Weight-bearing tendons in many species, including humans, chickens and horses, are prone to failure, in many cases without a discernible cause. The normal function of the tendon depends on the proper assembly of fibrils of type I collagen, the main structural component of the tendon. We studied the effect of in vitro culture, temperature (37 degrees C vs. 43 degrees C) and wounding on the expression of mRNAs for several collagen regulators, transforming growth factor beta (TGF(beta)), heat shock protein 47 (Hsp47) and connective tissue growth factor (CTGF), in chicken embryonic gastrocnemius tendon explants. The expression of mRNAs for TGF(beta) and Hsp47, a chaperone of collagen assembly, remained strong during the first day of in vitro culture, but then it decreased, slightly more at higher temperature. Additional injury in selected tendons had no significant effect on the levels of TGF(beta) and Hsp47 mRNAs. Likewise, the level of immunostained type I procollagen also decreased with the length of culture. The expression of CTGF gradually increased from 0 at the time of tendon removal with the duration of culture to strong after three days of culture when the expression of TGF(beta) and Hsp47 was low. We conclude that in vitro culture over the period of several days rather than an increase in temperature or additional wounding decreases the expression of TGF(beta), Hsp47 and type I procollagen and increases the expression of CTGF.

Regulation of formyl peptide receptor agonist affinity by reconstitution with arrestins and heterotrimeric G proteins.

Although heptahelical chemoattractant and chemokine receptors are known to play a significant role in the host immune response and the pathophysiology of disease, the molecular mechanisms and transient macroassemblies underlying their activation and regulation remain largely uncharacterized. We report herein real time analyses of molecular assemblies involving the formyl peptide receptor (FPR), a well described member of the chemoattractant subfamily of G protein-coupled receptors (GPCRs), with both arrestins and heterotrimeric G proteins. In our system, the ability to define and discriminate distinct, in vitro receptor complexes relies on quantitative differences in the dissociation rate of a fluorescent agonist as well as the guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) sensitivity of the complex, as recently described for FPR-G protein interactions. In the current study, we demonstrate a concentration- and time-dependent reconstitution of liganded, phosphorylated FPR with exogenous arrestin-2 and -3 to form a high agonist affinity, nucleotide-insensitive complex with EC(50) values of 0.5 and 0.9 microm, respectively. In contrast, neither arrestin-2 nor arrestin-3 altered the ligand dissociation kinetics of activated, nonphosphorylated FPR. Moreover, we demonstrated that the addition of G proteins was unable to alter the ligand dissociation kinetics or induce a GTP gamma S-sensitive state of the phosphorylated FPR. The properties of the phosphorylated FPR were entirely reversible upon treatment of the receptor preparation with phosphatase. These results represent to our knowledge the first report of the reconstitution of a detergent-solubilized, phosphorylated GPCR with arrestins and, furthermore, the first demonstration that phosphorylation of a nonvisual GPCR is capable of efficiently blocking G protein binding in the absence of arrestin. The significance of these results with respect to receptor desensitization and internalization are discussed.

Partial phosphorylation of the N-formyl peptide receptor inhibits G protein association independent of arrestin binding.

It is now well accepted that G protein-coupled receptors activated by agonist binding become targets for phosphorylation, leading to desensitization of the receptor. Using a series of phosphorylation deficient mutants of the N-formyl peptide receptor (FPR), we have explored the role of phosphorylation on the ability of the receptor to interact with G proteins and arrestins. Using a fluorometric assay in conjunction with solubilized receptors, we demonstrate that phosphorylation of the wild type FPR lowers its affinity for G protein, whereas mutant receptors lacking four potential phosphorylation sites retain their ability to couple to G protein. Phosphorylated mutant receptors lacking only two potential phosphorylation sites are again unable to couple to G protein. Furthermore, whereas stimulated wild type FPR in whole cells colocalizes with arrestin-2, and the solubilized, phosphorylated FPR binds arrestin-2, the stimulated receptors lacking four potential phosphorylation sites display no interaction with arrestin-2. However, the mutant receptors lacking only two potential phosphorylation sites are restored in their ability to bind and colocalize with arrestin-2. Thus, there is a submaximal threshold of FPR phosphorylation that simultaneously results in an inhibition of G protein binding and an induction of arrestin binding. These results are the first to demonstrate that less than maximal levels of receptor phosphorylation can block G protein binding, independent of arrestin binding. We therefore propose that phosphorylation alone may be sufficient to desensitize the FPR in vivo, raising the possibility that for certain G protein-coupled receptors, desensitization may not be the primary function of arrestin.

A rapid and strong laparoscopic-assisted gastropexy in dogs.

OBJECTIVE: To develop a technique for laparoscopic gastropexy in dogs and evaluate effects on stomach position and strength of the adhesion between the stomach and abdominal wall. ANIMALS: 8 healthy dogs. PROCEDURE: Dogs were anesthetized, and the abdomen was insufflated with carbon dioxide. A laparoscope was placed through a cannula inserted on the abdominal midline caudal to the umbilicus. Babcock forceps placed through a cannula inserted lateral to the right margin of the rectus abdominus muscle were used to exteriorize the pyloric antrum, a longitudinal incision was made through the serosa and muscular layer of the pyloric antrum, and the seromuscular layer of the pyloric antrum was sutured to the transversus abdominus muscle. After surgery, positive-contrast gastrography was used to evaluate stomach position and the onset of gastric emptying, and ultrasonography was used to assess stomach wall activity and mobility. Dogs were euthanatized 1 month after surgery, and tensile strength of the adhesion was tested. RESULTS: In all dogs, stomach position and the onset of gastric emptying were normal 25 days after surgery, and the pyloric antrum was firmly attached to the abdominal wall 30 days after surgery. Mean +/- SD ultimate load of the adhesion in tension was 106.5 +/- 45.6 N. CONCLUSIONS AND CLINICAL RELEVANCE: The laparoscopic gastropexy technique described in the present study could be performed quickly and easily by an experienced surgeon, resulted in a strong fibrous adhesion between the stomach and abdominal wall, and appeared to cause minimal stress to the dogs.

Validation of a finite element model of the Kirschner-Ehmer external skeletal fixation system.

OBJECTIVE: To determine the validity of finite element analysis (FEA) as a means of examining biomechanical properties of the Kirschner-Ehmer external skeletal fixation system. SAMPLE POPULATION: 10 paired tibiae harvested from skeletally mature dogs weighing between 30 and 38 kg immediately following euthanasia for reasons unrelated to musculoskeletal disease. PROCEDURE: A gap fracture was created in each bone; fragments were stabilized with 3 frame configurations (type I, type II, and type III), using enhanced-profile threaded pins. Each bone-frame construct was tested, using a materials testing machine in 3 modes of testing: axial compression (AC), mediolateral (ML) bending, and craniocaudal (CC) bending, for a total of 9 tests/bone. The elastic limit of the constructs was not exceeded during testing. Mean stiffness values were determined from load-displacement curves. A finite element model of each construct was created, using three-dimensional elastic beam elements, and stiffness values were calculated, using FEA. Correlations between experimental and FEA data then were determined. RESULTS: Significant differences in stiffness were seen among all 3 constructs in CC bending and AC, with stiffness increasing with construct complexity. No significant difference in ML bending stiffness was seen between type-II and type-III constructs; however, both were significantly stiffer than the type-I constructs. The experimental and FEA stiffness data were strongly correlated (AC, r = 0.994; ML bending, r = 0.998; CC bending, r = 0.985). CONCLUSIONS AND CLINICAL RELEVANCE: Strong correlations among experimental and FEA data indicate that FEA is a valid method of comparing stiffness of Kirschner-Ehmer external skeletal fixation constructs.

Type of milk consumed can influence plasma concentrations of fatty acids and minerals and body composition in infant and weanling pigs.

Two experiments using 42 crossbred neonatal pigs to compare the effects of caprine and bovine milk on growth, apparent nutrient digestibility and body composition were conducted. At age 72 h, pigs were removed from their dams and randomly divided into two groups, housed separately in stainless steel metabolism cages and were fed a predetermined amount (300 mL/kg body weight) of pasteurized, nonfortified whole, caprine or bovine milk. Body composition was determined using dual energy X-ray absorptiometry (DXA). In Experiment 1, 22 intact male pigs were used for a 31-d experimental period. There was no significant (P > 0.05) dietary effect on growth, apparent nutrient digestibility or body composition. Significant differences (P < 0.05), however, were observed in plasma of C 8:0, C 10:0 and C 12:0 concentrations. In Experiment 2, 20 pigs (10 intact males and 10 females) were used in a 2 x 2 factorial experiment for 52 d. Pigs fed caprine milk had higher (P < 0.05) plasma concentrations of C10:0 and C12:0 as well as Na, Mg and Zn than those fed bovine milk. At Day 52, pigs fed caprine milk had less body fat (P < 0.001) and higher (P < 0.06) bone mineral density than those fed bovine milk. Drymatter, N and total mineral intake of male pigs was higher (P < 0.05) than female pigs. Also, male pigs had higher (P < 0.05) plasma concentrations of C12:0 than females. This study demonstrates that the type of milk consumed can influence plasma concentrations of fatty acids, minerals and body composition in pigs.

MEK and ERK activation in ras-disabled RBL-2H3 mast cells and novel roles for geranylgeranylated and farnesylated proteins in Fc epsilonRI-mediated signaling.

Cross-linking the high affinity IgE receptor Fc epsilonRI of basophils and mast cells activates receptor-associated protein-tyrosine kinases and stimulates a signaling cascade leading to secretion, ruffling, spreading, and cytokine production. Previous evidence that the pan-prenylation inhibitor lovastatin blocks Ag-stimulated Ca2+ influx, secretion, and membrane/cytoskeletal responses implicated isoprenylated proteins in the Fc epsilonRI-coupled signaling cascade but could not distinguish between contributions of C15 (farnesylated) and C20 (geranylgeranylated) species. Here we establish concentrations of lovastatin and the farnesyl-specific inhibitor BZA-5B that inhibit the farnesylation and Ag-induced activation of Ras species in RBL-2H3 cells (H-Ras, K-RasA, and K-RasB). These inhibitors have little effect on tyrosine kinase activation, which initiates Fc epsilonRI signaling. Although Ras is disabled, only lovastatin substantially blocks Raf-1 activation, and neither inhibitor affects mitogen-activated protein kinase kinase/extracellular signal regulated kinase kinase (MEK) or ERK1/ERK2 activation. Thus, the pathway to Fc epsilonRI-mediated MEK/ERK and ERK activation can apparently bypass Ras and Raf-1. Predictably, only lovastatin inhibits Ag-induced ruffling, spreading, and secretion, previously linked to geranylgeranylated Rho and Rab family members. Additionally, only lovastatin inhibits phospholipase Cgamma-mediated inositol (1,4,5) trisphosphate production, sustained Ca2+ influx, and Ca2+-dependent IL-4 production, suggesting novel roles for geranylgeranylated (lovastatin-sensitive, BZA-5B-insensitive) proteins in Fc epsilonRI signal propagation. Remarkably, BZA-5B concentrations too low to inactivate Ras reduce the lag time to Ag-induced Ca2+ stores release and enhance secretion. These results link a non-Ras farnesylated protein(s) to the negative regulation of Ca2+ release from intracellular stores and secretion. We identified no clear role for Ras in Fc epsilonRI-coupled signaling but suggest its involvement in mast cell growth regulation based on the inhibition of cell proliferation by both BZA-5B and lovastatin.

Quantitative study of calcium uptake by tumorigenic bone (TE-85) and neuroblastoma x glioma (NG108-15) cells exposed to extremely-low-frequency (ELF) electric fields.

To verify the effect of cell culture state on frequency dependent increase in proliferation as well as Ca2+ flux across the plasma membrane, tumorigenic bone (TE-85) and neuroblastoma x glioma (NG108-15) cells cultured in the presence of fetal bovine serum (FBS) were exposed to capacitively coupled electric (CCEF) fields in the extremely low frequency (ELF) range of 10 to 18 Hz. [3H]Thymidine incorporation and 45Ca2+ uptake were used as endpoints. TE-85 cells cultured in the presence of 10% FBS did not exhibit a frequency dependent increase in proliferation in contrast to previous studies under growth arrested culture conditions, in which the cells were deprived of FBS. However, both TE-85 and NG108-15 cells had an increase in 45Ca2+ uptake in response to a 16 Hz 18.3 mV/cm CCEF. Fura-2 digital imaging microscopy was used to verify addition of 0.5 mM La3+ and 0.5 mM ionomycin as negative and positive controls, respectively. Imaging microscopy data was combined with 45Ca2+ incorporation results to quantify free intracellular calcium ([Ca2+]i) increase in response to CCEF exposure. TE-85 [Ca2+]i increased from 140 to 189-210 nM where as NG108-15 [Ca2+]i increased from 67 to 189-210 nM. These results suggested that serum deprivation may be a requirement for a frequency dependent increase in proliferation in TE-85 cells but is not necessary for the electric field induced increase in 45Ca2+ uptake in both TE-85 and NG108 cells. The present study also represents the first demonstration of increased 45Ca2+ uptake by neuroblastoma and/or glioma cells in response to an electric field exposure.

Prospective evaluation of ground reaction forces in dogs undergoing unilateral total hip replacement.

OBJECTIVE: To evaluate clinical and biomechanical gait variables in a group of dogs before and after (for 1 year) total hip replacement. ANIMALS: 16 dogs with degenerative joint disease of the coxofemoral joint secondary to hip dysplasia deemed candidates for total hip replacement. PROCEDURE: Before and at 1, 3, 6, and 12 months after surgery, each dog was trotted over a biomechanical force platform. Vertical force data evaluated for each stance phase of the treated and untreated hind limbs included peak force, impulse, and limb loading and unloading rates. Vertical peak and impulse data were also evaluated for the forelimbs. Measurements analyzed in the craniocaudal axis, divided into braking and propulsion phases, consisted of peak force and associated impulses. Also, orthopedic examination for each dog included subjective scoring for limb lameness at each evaluation period. RESULTS: Most ground reaction forces (GRF) were significantly lower before surgery for the proposed treated, compared with the proposed untreated, limb. This difference between limbs continued through postoperative month 1. Also at 1 month, some treated limb values were significantly lower than preoperative values. By 3 to 6 months, treated limb GRF increased so that no significant difference between limbs could be found. Vertical and craniocaudal propulsion impulse values were significantly higher in the treated than untreated limb from the 6-month evaluation period through the remainder of the study. Braking component of the craniocaudal axes measurements was unchanged throughout the study. CONCLUSIONS: GRF indicated that dogs of this study had significantly increased loading function of the treated hind limb by 6 months after unilateral total hip replacement. Data also indicated that some force was transferred from the untreated to treated hip over the study period. Loading rates also increased over the study period, indicating increased willingness to load the treated hip over time. Craniocaudal axis data indicated no improvement in braking forces with coxofemoral joint replacement, suggesting that the coxofemoral joint with degenerative joint disease did not have altered braking performance at a trotting gait. Comparison of subjective lameness scores and objective GRF indicated that visual grading of coxofemoral joint lameness is limited.

Characterization of the non-linear loading curve of rat skin.

A phenomenological model was developed to characterize the non-linear portion of stress-strain curves for skin tissue removed from laboratory rats and tangent modulus calculations were used to characterize the linear portion of the curves. Modified coefficient of determination calculations obtained from non-linear regression analysis showed that the model was able to fit the actual data above the 90% level. The model coefficients and tangent modulus values were used to statistical comparisons. Combination treatments of radiation, hyperthermia and surgical incision were used to analyze changes in the loading response of rat skin. Least significant difference statistical analysis (P < 0.10) of the model coefficients showed that radiation treatment affected skin stiffness only in tissue when surgical wounding was applied and that radiation-hyperthermia treatment affected skin stiffness only in tissue when surgical wounding was not applied. Hyperthermia applied with no radiation treatment did not affect skin stiffness. Surgical wounding resulted in decreasing skin stiffness.

Evaluation of limb symmetry indices, using ground reaction forces in healthy dogs.

Limb symmetry was evaluated by measuring ground reaction forces in 2 groups of normal-gaited dogs at a trot. Data were collected from 2 groups of 21 dogs trotted at dog/handler velocities of 1.25 to 1.55 m/s and 1.85 to 2.05 m/s, respectively. Of these dogs, 9 participated in both groups to allow comparison of data at both velocities. Additionally, 16 of the dogs in group 1 were measured in 2 directions of movement to determine whether directional dependence was present. Collected data were then applied to 3 described symmetry indices. Each index was easy to calculate, but all had limitations. A major limitation was variation in magnitude of ground reaction forces measured between the different axes and the effect of this variation on precision of the derived indices. Vertical ground forces provided the most consistent symmetry indices, in part because of their large magnitude. The indices indicated that no dog had perfect right-to-left symmetry during a trotting gait. Statistical differences were not found in any of the measurements of directional dependence. Likewise, comparing symmetry data in dogs trotted at both velocities indicated no significant differences in any axis. However, further analysis of the data revealed the actual amount that a variance attributable to right-left limb variation was negligible. Most of the variance was attributable to trial variation. Thus, the aforementioned indices, which use nonconsecutive footfall methods to evaluate limb symmetry, actually measure principally trial variation and not limb-to-limb variation.

Replacement of the medial collateral ligament with polypropylene mesh or a polyester suture in dogs.

The medial collateral ligament of one stifle in 20 adult dogs was excised and replaced with polypropylene mesh or a polyester suture. After 26 weeks, the fibrous tissue-prosthesis composites were evaluated clinically, morphologically, and biomechanically. Clinical lameness was not significantly different after 10 days. The polypropylene mesh reconstructions consistently had more fibrous tissue and greater collagenous ingrowth than the polyester suture reconstructions. There were four complications related to fixation of the polypropylene mesh prosthesis and one to the polyester suture. The polypropylene mesh reconstructions had greater stability and were biomechanically more similar to the natural ligaments than the polyester suture reconstructions. Although the results with polypropylene mesh were favorable, more challenging biomechanical testing and alternative anchoring techniques are required before polypropylene mesh can be recommended as a collateral ligament replacement in dogs.

Effects of freezing on mechanical properties of rat skin.

Two test specimens of skin were cut from the lateral aspect of each hind limb of 9 rats. Specimens were contiguous, thereby providing matched pairs. One specimen was immediately placed in liquid nitrogen for 5 minutes, then stored at -70 C and tested within 3 to 4 weeks. Within 5 minutes of harvest, the second specimen was used for immediate material testing. Basic engineering material tests were used to measure strength, loading response, and elastic and viscous properties. Each matched pair of tissues was used for the same procedure. Quasistatic uniaxial tensile tests were used to apply deformations to the test specimens, and resulting loads were recorded. Stress and strain were calculated from the recorded data, providing information on yield strength, ultimate strength, fracture strength, and loading response. Each matched pair of specimens represented 1 repetition; 6 repetitions were made of each observation. Statistical analysis indicated that tissue freezing significantly (P less than 0.05) increased fracture strength, but did not affect strength, ultimate strength, or loading response. Dynamic vibration response tests were used to find mechanical mobility of the specimens, thereby providing information on elastic and viscous behaviors, which were quantified by calculation of spring and damping coefficients, respectively. As before, 6 repetitions were used. Statistical analysis indicated that tissue freezing did not affect these coefficients.

Poult malabsorption syndrome. III. Skeletal lesions in market-age turkeys.

Skeletal deformities, a major contributing factor to economic losses in market-age turkeys, may be associated with the poult malabsorption syndrome. A study was performed to determine whether the naturally occurring malabsorption syndrome produces skeletal lesions in turkeys at market age. Poults were placed on litter on which poults had previously developed malabsorption. Exposed poults developed enteric disease with 21% mortality during the first 3 weeks. Controls had no enteric disease and no mortality. At the end of the study, 20-week-old exposed turkeys weighed 0.621 kg (5%) less than controls and had a higher incidence of angular limb deformities with significantly higher angulation scores. Exposed turkeys also had a greater incidence of rotated tibias and bowed tibias. The mechanical property of tibial shear strength was significantly lower in turkeys that had survived the earlier poult malabsorption syndrome.

A mathematical model to describe the nonlinear elastic properties of the gastrocnemius tendon of chickens.

A phenomenological model was developed to describe the nonlinear elastic behavior of the avian gastrocnemius tendon. Quasistatic uniaxial tensile tests were used to apply a deformation and resulting load on the tendon at a deformation rate of 5 mm/min. Plots of deformation versus load indicated a nonlinear loading response. By calculating engineering stress and engineering strain, the experimental data were normalized for tendon shape. The elastic response was determined from stress-strain curves and was found to vary with engineering strain. The response to the applied engineering strain could best be described by a mathematical model that combined a linear function and a nonlinear function. Three parameters in the model were developed to represent the nonlinear elastic behavior of the tendon, thereby allowing analysis of elasticity without prior knowledge of engineering strain. This procedure reduced the amount of data needed for the statistical analysis of nonlinear elasticity.