Whereas the important role of free radicals in diabetes-associated complications is well establis... more Whereas the important role of free radicals in diabetes-associated complications is well established, the contributions of the highly reactive oxidant peroxynitrite have not been properly explored. The present study used a pharmacological approach to evaluate the role of peroxynitrite in peripheral diabetic neuropathy. Control and STZ-diabetic mice were maintained with or without treatment with the potent peroxynitrite decomposition catalyst Fe(III) tetramesitylporphyrin octasulfonate (FeTMPS), at doses of 5 or 10 mg/kg/day in the drinking water for 3 weeks after an initial 3 weeks without treatment. Mice with a 6-week duration of diabetes developed clearly manifest motor (MNCV) and sensory nerve conduction velocity (SNCV) deficits, thermal hypoalgesia (paw withdrawal, tail-flick, and hot plate tests), mechanical hypoalgesia (tail pressure Randall-Sellito test), tactile allodynia (flexible von Frey filament test), and ~44% loss of intraepidermal nerve fibers. They also had increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in sciatic nerve, grey matter of the spinal cord, and dorsal root ganglion neurons. FeTMPS treatment alleviated or essentially corrected (at a dose of 10 mg/kg/day) MNCV and SNCV deficits, and was associated with less severe small sensory nerve fiber dysfunction and degeneration. Nitrotyrosine and poly(ADP-ribose) immunofluorescence in sciatic nerve, spinal cord, and dorsal root ganglion neurons in peroxynitrite decomposition catalyst-treated diabetic mice was markedly reduced. In conclusion, peroxynitrite contributes to large motor, large sensory, and small sensory fiber neuropathy in streptozotocin-diabetic mice. The findings provide rationale for development of potent peroxynitrite decomposition catalysts for the treatment of diabetic neuropathy.
Advances in Experimental Medicine and Biology, 2010
The mechanisms of contraction are intrinsically connected to -sarcomere mechanics during muscle a... more The mechanisms of contraction are intrinsically connected to -sarcomere mechanics during muscle activation and relaxation. This chapter presents two sets of experiments performed with (1) myofibrils, in which individual sarcomeres in series can be evaluated during contractions, and (2) mechanically isolated sarcomeres. When activated at optimal length (approximately 2.0-2.4 μm), myofibrils and sarcomeres produce similar forces. However, their dependence on length differs: sarcomeres in series in a myofibril are able to produce similar forces at distinct lengths, while isolated sarcomeres show a force-length relation that resembles that obtained in original studies performed with single muscle fibers. Although force in isolated sarcomeres is rapidly stabilized during activation, significant movements of A-band are present when the contraction is produced at optimal lengths, which leads to different dynamics in the two half-sarcomeres. A-band movements decrease linearly with increasing lengths between 2.6 and 3.6 μm. Myofibrils and sarcomeres represent reliable techniques to evaluate contractile mechanisms at the most basic level of muscle organization. However, they present different mechanical characteristics that must be taken into account when scientists evaluate mechanisms of contraction.
Evidence for the important role of the potent oxidant peroxynitrite in peripheral diabetic neurop... more Evidence for the important role of the potent oxidant peroxynitrite in peripheral diabetic neuropathy and neuropathic pain is emerging. This study evaluated the contribution of neuronal nitric oxide synthase (nNOS) to diabetes-induced nitrosative stress in peripheral nerve and dorsal root ganglia, and peripheral nerve dysfunction and degeneration. Control and nNOS-/mice were made diabetic with streptozotocin, and maintained for 6 weeks. Peroxynitrite injury was assessed by nitrotyrosine and poly(ADP-ribose) immunoreactivities. Peripheral diabetic neuropathy was evaluated by measurements of sciatic motor and hind-limb digital sensory nerve conduction velocities, thermal algesia, tactile allodynia, and intraepidermal nerve fiber density. Control nNOS-/mice displayed normal motor nerve conduction velocity and thermal response latency, whereas sensory nerve conduction velocity was slightly lower compared with non-diabetic wild-type mice, and tactile response threshold and intraepidermal nerve fiber density were reduced by 47 and 38%, respectively. Both diabetic wild-type and nNOS-/mice displayed enhanced nitrosative stress in peripheral nerve. In contrast to diabetic wild-type mice, diabetic nNOS-/mice had near normal nitrotyrosine and poly(ADP-ribose) immunofluorescence in dorsal root ganglia. Both diabetic wild-type and nNOS-/mice developed motor and sensory nerve conduction velocity deficits and thermal hypoalgesia although nNOS gene deficiency slightly reduced severity of the three disorders. Tactile response thresholds were similarly decreased in control and diabetic nNOS-/mice compared with non-diabetic wild-type mice. Intraepidermal nerve fiber density was lower by 27% in diabetic nNOS-/mice compared with the corresponding nondiabetic group, and by 20% in diabetic nNOS-/mice compared with diabetic wild-type mice. In conclusion, nNOS is required for maintaining the normal peripheral nerve function and small sensory nerve fibre innervation. nNOS gene deficiency does not protect from development of nerve conduction deficit, sensory neuropathy and intraepidermal nerve fiber loss.
Background: Contractile properties of myofibrils from the myocardium and diaphragm in chronic hea... more Background: Contractile properties of myofibrils from the myocardium and diaphragm in chronic heart failure are not well understood. We investigated myofibrils in a knockout (KO) mouse model with cardiac-specific deletion of arginyl-tRNA-protein transferase (α-MHCAte1), which presents dilated cardiomyopathy and heart failure. Objective: The aim of this study was to test the hypothesis that chronic heart failure in α-MHCAte1 mice is associated with abnormal contractile properties of the heart and diaphragm. Methods: We used a newly developed system of atomic force cantilevers (AFC) to compare myofibrils from α-MHCAte1 and age-matched wild type mice (WT). Myofibrils from the myocardium and the diaphragm were attached to the AFC used for force measurements during activation/deactivation cycles at different sarcomere lengths. Results: In the heart, α-MHCAte1 myofibrils presented a reduced force during full activation (89 ± 9 nN/μm 2) when compared to WT (132 ± 11 nN/μm 2), and the decrease was not influenced by sarcomere length. These myofibrils presented similar kinetics of force development (K act), redevelopment (K tr), and relaxation (K rel). In the diaphragm, α-MHCAte1 myofibrils presented an increased force during full activation (209 ± 31 nN/μm 2) when compared to WT (123 ± 20 nN/μm 2). Diaphragm myofibrils of α-MHCAte1 and WT presented similar K act , but α-MHCAte1 myofibrils presented a faster K rel (6.11 ± 0.41 s −1 vs 4.63 ± 0.41 s −1). Conclusion: Contrary to our working hypothesis, diaphragm myofibrils from α-MHCAte1 mice produced an increased force compared to myofibrils from WT. These results suggest a potential compensatory mechanism by which the diaphragm works under loading conditions in the α-MHCAte1 chronic heart failure model.
Evidence for important role of poly(ADP-ribose) polymerase (PARP) activation in diabetic complica... more Evidence for important role of poly(ADP-ribose) polymerase (PARP) activation in diabetic complications is emerging. This study evaluated the role for PARP in rat and mouse models of advanced diabetic neuropathy. The orally active PARP inhibitor 10-(4-methyl-piperazin-1ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one(GPI-15427, formulated as mesilate salt, 30 mg kg −1 d −1 in the drinking water, for 10 weeks after first 2 weeks without treatment) at least partially prevented PARP activation in peripheral nerve and DRG neurons, as well as thermal hypoalgesia, mechanical hyperalgesia, tactile allodynia, exaggerated response to formalin, and, the most important, intraepidermal nerve fiber degeneration in streptozotocin-diabetic rats. These findings are consistent with the lack of small sensory nerve fiber dysfunction in diabetic PARP−/− mice. Furthermore, whereas diabetic PARP+/+ mice displayed ~ 46% intraepidermal nerve fiber loss, diabetic PARP−/− preserved completely normal intraepidermal nerve fiber density. In conclusion, PARP activation is an important contributor to intraepidermal nerve fiber degeneration and functional changes associated with advanced Type 1 diabetic neuropathy. The results support the rationale for development of potent and low toxic PARP inhibitors and PARP inhibitorcontaining combination therapies. Keywords intraepidermal nerve fiber loss; mechanical hyperalgesia; mechanical hypoalgesia; neuropathic pain; oxidative-nitrosative stress; poly(ADP-ribose) polymerase; tactile allodynia; thermal hypoalgesia Evidence for a fundamental role of poly(ADP-ribose) polymerase (PARP) (1) in diabetic complications including endothelial (2) and myocardial (3) dysfunction, peripheral (PDN) (4,5) and autonomic (6) neuropathy, retinopathy (7,8), and nephropathy (9,10), is emerging. PARP activation manifest by accumulation of poly(ADP-ribose) polymer has been observed
Whereas an important role of free radicals and oxidants in peripheral diabetic neuropathy is well... more Whereas an important role of free radicals and oxidants in peripheral diabetic neuropathy is well established, the contribution of nitrosative stress and, in particular, of the highly reactive oxidant peroxynitrite, has not been properly explored. Our previous findings implicate peroxynitrite in diabetes-associated motor and sensory nerve conduction deficits and peripheral nerve energy deficiency and poly(ADP-ribose) polymerase activation associated with Type 1 diabetes. In this study the role of nitrosative stress in diabetic sensory neuropathy is evaluated. The peroxynitrite decomposition catalyst Fe(III) tetrakis-2-(N-triethylene glycol monomethyl ether)pyridyl porphyrin (FP15) was administered to control and streptozotocin (STZ)-diabetic mice at the dose of 5 mg kg −1 day −1 (FP15), for 3 weeks after initial 3 weeks without treatment. Mice with 6-week duration of diabetes developed clearly manifest thermal hypoalgesia (paw withdrawal, tail-flick, and hot plate tests), mechanical hypoalgesia (tail pressure Randall-Sellito test), tactile allodynia (flexible von Frey filament test), and ~38% loss of intraepidermal nerve fibers. They also had increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in the sciatic nerve, grey matter of spinal cord, and dorsal root ganglion neurons. FP15 treatment was associated with alleviation of thermal and mechanical hypoalgesia. Tactile response threshold tended to increase in response to peroxynitrite decomposition catalyst treatment, but still remained ~59% lower compared with non-diabetic controls. Intraepidermal nerve fiber density was 25% higher in FP15-treated than in untreated diabetic rats, but the difference between two groups did not achieve statistical significance (p=0.054). Nitrotyrosine and poly(ADP-ribose) immunofluorescence in sciatic nerve, spinal cord, and dorsal root ganglion neurons of peroxynitrite decomposition catalyst-treated diabetic mice were markedly reduced. In conclusion, nitrosative stress plays an important role in sensory neuropathy associated with Type 1 diabetes. The findings provide rationale for further studies of peroxynitrite decomposition catalysts in a long-term diabetic model.
Aims/hypothesis Evidence for the importance of peroxynitrite, a product of superoxide anion radic... more Aims/hypothesis Evidence for the importance of peroxynitrite, a product of superoxide anion radical reaction with nitric oxide, in peripheral diabetic neuropathy is emerging. The role of specific nitric oxide synthase isoforms in diabetesassociated nitrosative stress and nerve fibre dysfunction and degeneration remains unknown. This study evaluated the contribution of inducible nitric oxide synthase (iNOS) to peroxynitrite injury to peripheral nerve and dorsal root ganglia and development of peripheral diabetic neuropathy. Methods Control mice and mice with iNos (also known as Nos2) gene deficiency (iNos −/−) were made diabetic with streptozotocin, and maintained for 6 weeks. Peroxynitrite injury was assessed by nitrotyrosine and poly(ADP-ribose) accumulation (immunohistochemistry). Thermal algesia was evaluated by paw withdrawal, tail-flick and hot plate tests, mechanical algesia by the Randall-Selitto test, and tactile allodynia by a von Frey filament test. Results Diabetic wild-type mice displayed peroxynitrite injury in peripheral nerve and dorsal root ganglion neurons. They also developed motor and sensory nerve conduction velocity deficits, thermal and mechanical hypoalgesia, tactile allodynia and ∼36% loss of intraepidermal nerve fibres. Diabetic iNos −/− mice did not display nitrotyrosine and poly(ADP-ribose) accumulation in peripheral nerve, but were not protected from nitrosative stress in dorsal root ganglia. Despite this latter circumstance, diabetic iNos −/− mice preserved normal nerve conduction velocities. Small-fibre sensory neuropathy was also less severe in diabetic iNos −/− than in wild-type mice. Conclusions/interpretation iNOS plays a key role in peroxynitrite injury to peripheral nerve, and functional and structural changes of diabetic neuropathy. Nitrosative stress in axons and Schwann cells, rather than dorsal root ganglion neurons, underlies peripheral nerve dysfunction and degeneration. Keywords iNOS. Nerve conduction. Nitrosative stress. Peripheral diabetic neuropathy. Tactile allodynia. Thermal algesia Abbreviations DAB 3,3′-diaminobenzidine DRG dorsal root ganglion INFD intraepidermal nerve fibre density iNOS inducible nitric oxide synthase MNCV motor nerve conduction velocity NT nitrotyrosine PAR poly(ADP-ribose) PARP poly(ADP-ribose) polymerase PDN peripheral diabetic neuropathy PGP 9.5 protein gene product 9.5 SNCV sensory nerve conduction velocity STZ streptozotocin TBS TRIS-buffered saline
OBJECTIVE-Subjects with dietary obesity and pre-diabetes have an increased risk for developing bo... more OBJECTIVE-Subjects with dietary obesity and pre-diabetes have an increased risk for developing both nerve conduction slowing and small sensory fiber neuropathy. Animal models of this type of neuropathy have not been described. This study evaluated neuropathic changes and their amenability to dietary and pharmacological interventions in mice fed a high-fat diet (HFD), a model of pre-diabetes and alimentary obesity. RESEARCH DESIGN AND METHODS-Female C57BL6/J mice were fed normal diets or HFDs for 16 weeks. RESULTS-HFD-fed mice developed obesity, increased plasma FFA and insulin concentrations, and impaired glucose tolerance. They also had motor and sensory nerve conduction deficits, tactile allodynia, and thermal hypoalgesia in the absence of intraepidermal nerve fiber loss or axonal atrophy. Despite the absence of overt hyperglycemia, the mice displayed augmented sorbitol pathway activity in the peripheral nerve, as well as 4-hydroxynonenal adduct nitrotyrosine and poly(ADP-ribose) accumulation and 12/15-lipoxygenase overexpression in peripheral nerve and dorsal root ganglion neurons. A 6-week feeding with normal chow after 16 weeks on HFD alleviated tactile allodynia and essentially corrected thermal hypoalgesia and sensory nerve conduction deficit without affecting motor nerve conduction slowing. Normal chow containing the aldose reductase inhibitor fidarestat (16 mg ⅐ kg Ϫ1 ⅐ day Ϫ1) corrected all functional changes of HFD-induced neuropathy. CONCLUSIONS-Similar to human subjects with pre-diabetes and obesity, HFD-fed mice develop peripheral nerve functional, but not structural, abnormalities and, therefore, are a suitable model for evaluating dietary and pharmacological approaches to halt progression and reverse diabetic neuropathy at the earliest stage of the disease.
We demonstrate an amplitude-based bending/displacement sensor that uses a plastic photonic bandga... more We demonstrate an amplitude-based bending/displacement sensor that uses a plastic photonic bandgap Bragg fiber with one end coated with a silver layer. The reflection intensity of the Bragg fiber is characterized in response to different displacements (or bending curvatures). We note that the Bragg reflector of the fiber acts as an efficient mode stripper for the wavelengths near the edge of the fiber bandgap, which makes the sensor extremely sensitive to bending or displacements at these wavelengths. Besides, by comparison of the Bragg fiber sensor to a sensor based on a standard multimode fiber with similar outer diameter and length, we find that the Bragg fiber sensor is more sensitive to bending due to the presence of a mode stripper in the form of a multilayer reflector. Experimental results show that the minimum detection limit of the Bragg fiber sensor can be as small as 3 μm for displacement sensing.
In this study, we have tried to understand why the left ventricle (LV) is not a homogeneous spher... more In this study, we have tried to understand why the left ventricle (LV) is not a homogeneous sphere. An experimental model of a spherical ventricle was developed. The chamber was configured as a mathematical model, and the wall properties were represented by isolated cardiac muscles. The stroke work of the spherical LV when modelling different types of inhomogeneity in the wall structure was investigated. It was found that the emergence of even slight inhomogeneity in a spherical ventricle inevitably results in a diminution of pump function. It was concluded that at a given level of the myocardial contractility, a homogeneous spherical LV would not have any functional reserve, ie no ability to maintain pump function in case of additional load. Functional reserve can be achieved only with a certain degree of inhomogeneity. Thus, inhomogeneity in the normal left ventricular wall structure constitutes a strategic functional reserve that is absent in a homogeneous spherical ventricle.
The goal of this study was to develop a system to experiment with sarcomeres mechanically isolate... more The goal of this study was to develop a system to experiment with sarcomeres mechanically isolated from skeletal muscles. Single myofibrils from rabbit psoas were transferred into a temperature-controlled (22°C or 15°C) experimental chamber, and sarcomeres were isolated using precalibrated glass microneedles that were pierced externally, adjacent to the Z-lines. The force produced during activation was measured by tracking the displacement of the microneedles, and the sarcomere and half-sarcomere changes were measured by continuously tracking the Z-lines and A-bands position during the experiments. Sarcomeres produced a stress (force/cross-sectional area) of 112.75 ± 4.96 nN/μm2 (15°C) and 128.47 ± 5.58 nN/μm2 (22°C) at lengths between 2.0 μm and 2.4 μm. The descending limb was fitted with linear regression for length between 2.4 μm and 3.5 μm, which provided an abscissa extrapolating to 3.87 μm. The force-length relation was remarkably similar to a theoretical curve based on the de...
When a stretch is imposed to activated muscles, there is a residual force enhancement that persis... more When a stretch is imposed to activated muscles, there is a residual force enhancement that persists after the stretch; the force is higher than that produced during an isometric contraction in the corresponding length. The mechanisms behind the force enhancement remain elusive, and there is disagreement if it represents a sarcomeric property, or if it is associated with length nonuniformities among sarcomeres and half-sarcomeres. The purpose of this study was to investigate the effects of stretch on single sarcomeres and myofibrils with predetermined numbers of sarcomeres ( n = 2, 3. . . , 8) isolated from the rabbit psoas muscle. Sarcomeres were attached between two precalibrated microneedles for force measurements, and images of the preparations were projected onto a linear photodiode array for measurements of half-sarcomere length (SL). Fully activated sarcomeres were subjected to a stretch (5–10% of initial SL, at a speed of 0.3 μm·s−1·SL−1) after which they were maintained isom...
Whereas the important role of free radicals in diabetes-associated complications is well establis... more Whereas the important role of free radicals in diabetes-associated complications is well established, the contributions of the highly reactive oxidant peroxynitrite have not been properly explored. The present study used a pharmacological approach to evaluate the role of peroxynitrite in peripheral diabetic neuropathy. Control and STZ-diabetic mice were maintained with or without treatment with the potent peroxynitrite decomposition catalyst Fe(III) tetramesitylporphyrin octasulfonate (FeTMPS), at doses of 5 or 10 mg/kg/day in the drinking water for 3 weeks after an initial 3 weeks without treatment. Mice with a 6-week duration of diabetes developed clearly manifest motor (MNCV) and sensory nerve conduction velocity (SNCV) deficits, thermal hypoalgesia (paw withdrawal, tail-flick, and hot plate tests), mechanical hypoalgesia (tail pressure Randall-Sellito test), tactile allodynia (flexible von Frey filament test), and ~44% loss of intraepidermal nerve fibers. They also had increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in sciatic nerve, grey matter of the spinal cord, and dorsal root ganglion neurons. FeTMPS treatment alleviated or essentially corrected (at a dose of 10 mg/kg/day) MNCV and SNCV deficits, and was associated with less severe small sensory nerve fiber dysfunction and degeneration. Nitrotyrosine and poly(ADP-ribose) immunofluorescence in sciatic nerve, spinal cord, and dorsal root ganglion neurons in peroxynitrite decomposition catalyst-treated diabetic mice was markedly reduced. In conclusion, peroxynitrite contributes to large motor, large sensory, and small sensory fiber neuropathy in streptozotocin-diabetic mice. The findings provide rationale for development of potent peroxynitrite decomposition catalysts for the treatment of diabetic neuropathy.
Advances in Experimental Medicine and Biology, 2010
The mechanisms of contraction are intrinsically connected to -sarcomere mechanics during muscle a... more The mechanisms of contraction are intrinsically connected to -sarcomere mechanics during muscle activation and relaxation. This chapter presents two sets of experiments performed with (1) myofibrils, in which individual sarcomeres in series can be evaluated during contractions, and (2) mechanically isolated sarcomeres. When activated at optimal length (approximately 2.0-2.4 μm), myofibrils and sarcomeres produce similar forces. However, their dependence on length differs: sarcomeres in series in a myofibril are able to produce similar forces at distinct lengths, while isolated sarcomeres show a force-length relation that resembles that obtained in original studies performed with single muscle fibers. Although force in isolated sarcomeres is rapidly stabilized during activation, significant movements of A-band are present when the contraction is produced at optimal lengths, which leads to different dynamics in the two half-sarcomeres. A-band movements decrease linearly with increasing lengths between 2.6 and 3.6 μm. Myofibrils and sarcomeres represent reliable techniques to evaluate contractile mechanisms at the most basic level of muscle organization. However, they present different mechanical characteristics that must be taken into account when scientists evaluate mechanisms of contraction.
Evidence for the important role of the potent oxidant peroxynitrite in peripheral diabetic neurop... more Evidence for the important role of the potent oxidant peroxynitrite in peripheral diabetic neuropathy and neuropathic pain is emerging. This study evaluated the contribution of neuronal nitric oxide synthase (nNOS) to diabetes-induced nitrosative stress in peripheral nerve and dorsal root ganglia, and peripheral nerve dysfunction and degeneration. Control and nNOS-/mice were made diabetic with streptozotocin, and maintained for 6 weeks. Peroxynitrite injury was assessed by nitrotyrosine and poly(ADP-ribose) immunoreactivities. Peripheral diabetic neuropathy was evaluated by measurements of sciatic motor and hind-limb digital sensory nerve conduction velocities, thermal algesia, tactile allodynia, and intraepidermal nerve fiber density. Control nNOS-/mice displayed normal motor nerve conduction velocity and thermal response latency, whereas sensory nerve conduction velocity was slightly lower compared with non-diabetic wild-type mice, and tactile response threshold and intraepidermal nerve fiber density were reduced by 47 and 38%, respectively. Both diabetic wild-type and nNOS-/mice displayed enhanced nitrosative stress in peripheral nerve. In contrast to diabetic wild-type mice, diabetic nNOS-/mice had near normal nitrotyrosine and poly(ADP-ribose) immunofluorescence in dorsal root ganglia. Both diabetic wild-type and nNOS-/mice developed motor and sensory nerve conduction velocity deficits and thermal hypoalgesia although nNOS gene deficiency slightly reduced severity of the three disorders. Tactile response thresholds were similarly decreased in control and diabetic nNOS-/mice compared with non-diabetic wild-type mice. Intraepidermal nerve fiber density was lower by 27% in diabetic nNOS-/mice compared with the corresponding nondiabetic group, and by 20% in diabetic nNOS-/mice compared with diabetic wild-type mice. In conclusion, nNOS is required for maintaining the normal peripheral nerve function and small sensory nerve fibre innervation. nNOS gene deficiency does not protect from development of nerve conduction deficit, sensory neuropathy and intraepidermal nerve fiber loss.
Background: Contractile properties of myofibrils from the myocardium and diaphragm in chronic hea... more Background: Contractile properties of myofibrils from the myocardium and diaphragm in chronic heart failure are not well understood. We investigated myofibrils in a knockout (KO) mouse model with cardiac-specific deletion of arginyl-tRNA-protein transferase (α-MHCAte1), which presents dilated cardiomyopathy and heart failure. Objective: The aim of this study was to test the hypothesis that chronic heart failure in α-MHCAte1 mice is associated with abnormal contractile properties of the heart and diaphragm. Methods: We used a newly developed system of atomic force cantilevers (AFC) to compare myofibrils from α-MHCAte1 and age-matched wild type mice (WT). Myofibrils from the myocardium and the diaphragm were attached to the AFC used for force measurements during activation/deactivation cycles at different sarcomere lengths. Results: In the heart, α-MHCAte1 myofibrils presented a reduced force during full activation (89 ± 9 nN/μm 2) when compared to WT (132 ± 11 nN/μm 2), and the decrease was not influenced by sarcomere length. These myofibrils presented similar kinetics of force development (K act), redevelopment (K tr), and relaxation (K rel). In the diaphragm, α-MHCAte1 myofibrils presented an increased force during full activation (209 ± 31 nN/μm 2) when compared to WT (123 ± 20 nN/μm 2). Diaphragm myofibrils of α-MHCAte1 and WT presented similar K act , but α-MHCAte1 myofibrils presented a faster K rel (6.11 ± 0.41 s −1 vs 4.63 ± 0.41 s −1). Conclusion: Contrary to our working hypothesis, diaphragm myofibrils from α-MHCAte1 mice produced an increased force compared to myofibrils from WT. These results suggest a potential compensatory mechanism by which the diaphragm works under loading conditions in the α-MHCAte1 chronic heart failure model.
Evidence for important role of poly(ADP-ribose) polymerase (PARP) activation in diabetic complica... more Evidence for important role of poly(ADP-ribose) polymerase (PARP) activation in diabetic complications is emerging. This study evaluated the role for PARP in rat and mouse models of advanced diabetic neuropathy. The orally active PARP inhibitor 10-(4-methyl-piperazin-1ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one(GPI-15427, formulated as mesilate salt, 30 mg kg −1 d −1 in the drinking water, for 10 weeks after first 2 weeks without treatment) at least partially prevented PARP activation in peripheral nerve and DRG neurons, as well as thermal hypoalgesia, mechanical hyperalgesia, tactile allodynia, exaggerated response to formalin, and, the most important, intraepidermal nerve fiber degeneration in streptozotocin-diabetic rats. These findings are consistent with the lack of small sensory nerve fiber dysfunction in diabetic PARP−/− mice. Furthermore, whereas diabetic PARP+/+ mice displayed ~ 46% intraepidermal nerve fiber loss, diabetic PARP−/− preserved completely normal intraepidermal nerve fiber density. In conclusion, PARP activation is an important contributor to intraepidermal nerve fiber degeneration and functional changes associated with advanced Type 1 diabetic neuropathy. The results support the rationale for development of potent and low toxic PARP inhibitors and PARP inhibitorcontaining combination therapies. Keywords intraepidermal nerve fiber loss; mechanical hyperalgesia; mechanical hypoalgesia; neuropathic pain; oxidative-nitrosative stress; poly(ADP-ribose) polymerase; tactile allodynia; thermal hypoalgesia Evidence for a fundamental role of poly(ADP-ribose) polymerase (PARP) (1) in diabetic complications including endothelial (2) and myocardial (3) dysfunction, peripheral (PDN) (4,5) and autonomic (6) neuropathy, retinopathy (7,8), and nephropathy (9,10), is emerging. PARP activation manifest by accumulation of poly(ADP-ribose) polymer has been observed
Whereas an important role of free radicals and oxidants in peripheral diabetic neuropathy is well... more Whereas an important role of free radicals and oxidants in peripheral diabetic neuropathy is well established, the contribution of nitrosative stress and, in particular, of the highly reactive oxidant peroxynitrite, has not been properly explored. Our previous findings implicate peroxynitrite in diabetes-associated motor and sensory nerve conduction deficits and peripheral nerve energy deficiency and poly(ADP-ribose) polymerase activation associated with Type 1 diabetes. In this study the role of nitrosative stress in diabetic sensory neuropathy is evaluated. The peroxynitrite decomposition catalyst Fe(III) tetrakis-2-(N-triethylene glycol monomethyl ether)pyridyl porphyrin (FP15) was administered to control and streptozotocin (STZ)-diabetic mice at the dose of 5 mg kg −1 day −1 (FP15), for 3 weeks after initial 3 weeks without treatment. Mice with 6-week duration of diabetes developed clearly manifest thermal hypoalgesia (paw withdrawal, tail-flick, and hot plate tests), mechanical hypoalgesia (tail pressure Randall-Sellito test), tactile allodynia (flexible von Frey filament test), and ~38% loss of intraepidermal nerve fibers. They also had increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in the sciatic nerve, grey matter of spinal cord, and dorsal root ganglion neurons. FP15 treatment was associated with alleviation of thermal and mechanical hypoalgesia. Tactile response threshold tended to increase in response to peroxynitrite decomposition catalyst treatment, but still remained ~59% lower compared with non-diabetic controls. Intraepidermal nerve fiber density was 25% higher in FP15-treated than in untreated diabetic rats, but the difference between two groups did not achieve statistical significance (p=0.054). Nitrotyrosine and poly(ADP-ribose) immunofluorescence in sciatic nerve, spinal cord, and dorsal root ganglion neurons of peroxynitrite decomposition catalyst-treated diabetic mice were markedly reduced. In conclusion, nitrosative stress plays an important role in sensory neuropathy associated with Type 1 diabetes. The findings provide rationale for further studies of peroxynitrite decomposition catalysts in a long-term diabetic model.
Aims/hypothesis Evidence for the importance of peroxynitrite, a product of superoxide anion radic... more Aims/hypothesis Evidence for the importance of peroxynitrite, a product of superoxide anion radical reaction with nitric oxide, in peripheral diabetic neuropathy is emerging. The role of specific nitric oxide synthase isoforms in diabetesassociated nitrosative stress and nerve fibre dysfunction and degeneration remains unknown. This study evaluated the contribution of inducible nitric oxide synthase (iNOS) to peroxynitrite injury to peripheral nerve and dorsal root ganglia and development of peripheral diabetic neuropathy. Methods Control mice and mice with iNos (also known as Nos2) gene deficiency (iNos −/−) were made diabetic with streptozotocin, and maintained for 6 weeks. Peroxynitrite injury was assessed by nitrotyrosine and poly(ADP-ribose) accumulation (immunohistochemistry). Thermal algesia was evaluated by paw withdrawal, tail-flick and hot plate tests, mechanical algesia by the Randall-Selitto test, and tactile allodynia by a von Frey filament test. Results Diabetic wild-type mice displayed peroxynitrite injury in peripheral nerve and dorsal root ganglion neurons. They also developed motor and sensory nerve conduction velocity deficits, thermal and mechanical hypoalgesia, tactile allodynia and ∼36% loss of intraepidermal nerve fibres. Diabetic iNos −/− mice did not display nitrotyrosine and poly(ADP-ribose) accumulation in peripheral nerve, but were not protected from nitrosative stress in dorsal root ganglia. Despite this latter circumstance, diabetic iNos −/− mice preserved normal nerve conduction velocities. Small-fibre sensory neuropathy was also less severe in diabetic iNos −/− than in wild-type mice. Conclusions/interpretation iNOS plays a key role in peroxynitrite injury to peripheral nerve, and functional and structural changes of diabetic neuropathy. Nitrosative stress in axons and Schwann cells, rather than dorsal root ganglion neurons, underlies peripheral nerve dysfunction and degeneration. Keywords iNOS. Nerve conduction. Nitrosative stress. Peripheral diabetic neuropathy. Tactile allodynia. Thermal algesia Abbreviations DAB 3,3′-diaminobenzidine DRG dorsal root ganglion INFD intraepidermal nerve fibre density iNOS inducible nitric oxide synthase MNCV motor nerve conduction velocity NT nitrotyrosine PAR poly(ADP-ribose) PARP poly(ADP-ribose) polymerase PDN peripheral diabetic neuropathy PGP 9.5 protein gene product 9.5 SNCV sensory nerve conduction velocity STZ streptozotocin TBS TRIS-buffered saline
OBJECTIVE-Subjects with dietary obesity and pre-diabetes have an increased risk for developing bo... more OBJECTIVE-Subjects with dietary obesity and pre-diabetes have an increased risk for developing both nerve conduction slowing and small sensory fiber neuropathy. Animal models of this type of neuropathy have not been described. This study evaluated neuropathic changes and their amenability to dietary and pharmacological interventions in mice fed a high-fat diet (HFD), a model of pre-diabetes and alimentary obesity. RESEARCH DESIGN AND METHODS-Female C57BL6/J mice were fed normal diets or HFDs for 16 weeks. RESULTS-HFD-fed mice developed obesity, increased plasma FFA and insulin concentrations, and impaired glucose tolerance. They also had motor and sensory nerve conduction deficits, tactile allodynia, and thermal hypoalgesia in the absence of intraepidermal nerve fiber loss or axonal atrophy. Despite the absence of overt hyperglycemia, the mice displayed augmented sorbitol pathway activity in the peripheral nerve, as well as 4-hydroxynonenal adduct nitrotyrosine and poly(ADP-ribose) accumulation and 12/15-lipoxygenase overexpression in peripheral nerve and dorsal root ganglion neurons. A 6-week feeding with normal chow after 16 weeks on HFD alleviated tactile allodynia and essentially corrected thermal hypoalgesia and sensory nerve conduction deficit without affecting motor nerve conduction slowing. Normal chow containing the aldose reductase inhibitor fidarestat (16 mg ⅐ kg Ϫ1 ⅐ day Ϫ1) corrected all functional changes of HFD-induced neuropathy. CONCLUSIONS-Similar to human subjects with pre-diabetes and obesity, HFD-fed mice develop peripheral nerve functional, but not structural, abnormalities and, therefore, are a suitable model for evaluating dietary and pharmacological approaches to halt progression and reverse diabetic neuropathy at the earliest stage of the disease.
We demonstrate an amplitude-based bending/displacement sensor that uses a plastic photonic bandga... more We demonstrate an amplitude-based bending/displacement sensor that uses a plastic photonic bandgap Bragg fiber with one end coated with a silver layer. The reflection intensity of the Bragg fiber is characterized in response to different displacements (or bending curvatures). We note that the Bragg reflector of the fiber acts as an efficient mode stripper for the wavelengths near the edge of the fiber bandgap, which makes the sensor extremely sensitive to bending or displacements at these wavelengths. Besides, by comparison of the Bragg fiber sensor to a sensor based on a standard multimode fiber with similar outer diameter and length, we find that the Bragg fiber sensor is more sensitive to bending due to the presence of a mode stripper in the form of a multilayer reflector. Experimental results show that the minimum detection limit of the Bragg fiber sensor can be as small as 3 μm for displacement sensing.
In this study, we have tried to understand why the left ventricle (LV) is not a homogeneous spher... more In this study, we have tried to understand why the left ventricle (LV) is not a homogeneous sphere. An experimental model of a spherical ventricle was developed. The chamber was configured as a mathematical model, and the wall properties were represented by isolated cardiac muscles. The stroke work of the spherical LV when modelling different types of inhomogeneity in the wall structure was investigated. It was found that the emergence of even slight inhomogeneity in a spherical ventricle inevitably results in a diminution of pump function. It was concluded that at a given level of the myocardial contractility, a homogeneous spherical LV would not have any functional reserve, ie no ability to maintain pump function in case of additional load. Functional reserve can be achieved only with a certain degree of inhomogeneity. Thus, inhomogeneity in the normal left ventricular wall structure constitutes a strategic functional reserve that is absent in a homogeneous spherical ventricle.
The goal of this study was to develop a system to experiment with sarcomeres mechanically isolate... more The goal of this study was to develop a system to experiment with sarcomeres mechanically isolated from skeletal muscles. Single myofibrils from rabbit psoas were transferred into a temperature-controlled (22°C or 15°C) experimental chamber, and sarcomeres were isolated using precalibrated glass microneedles that were pierced externally, adjacent to the Z-lines. The force produced during activation was measured by tracking the displacement of the microneedles, and the sarcomere and half-sarcomere changes were measured by continuously tracking the Z-lines and A-bands position during the experiments. Sarcomeres produced a stress (force/cross-sectional area) of 112.75 ± 4.96 nN/μm2 (15°C) and 128.47 ± 5.58 nN/μm2 (22°C) at lengths between 2.0 μm and 2.4 μm. The descending limb was fitted with linear regression for length between 2.4 μm and 3.5 μm, which provided an abscissa extrapolating to 3.87 μm. The force-length relation was remarkably similar to a theoretical curve based on the de...
When a stretch is imposed to activated muscles, there is a residual force enhancement that persis... more When a stretch is imposed to activated muscles, there is a residual force enhancement that persists after the stretch; the force is higher than that produced during an isometric contraction in the corresponding length. The mechanisms behind the force enhancement remain elusive, and there is disagreement if it represents a sarcomeric property, or if it is associated with length nonuniformities among sarcomeres and half-sarcomeres. The purpose of this study was to investigate the effects of stretch on single sarcomeres and myofibrils with predetermined numbers of sarcomeres ( n = 2, 3. . . , 8) isolated from the rabbit psoas muscle. Sarcomeres were attached between two precalibrated microneedles for force measurements, and images of the preparations were projected onto a linear photodiode array for measurements of half-sarcomere length (SL). Fully activated sarcomeres were subjected to a stretch (5–10% of initial SL, at a speed of 0.3 μm·s−1·SL−1) after which they were maintained isom...
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Papers by Ivan Pavlov