Lisa Stehno-Bittel

Laser photostimulation of collagen production in healing rabbit Achilles tendons

Low energy laser photostimulation at certain wavelengths can enhance tissue repair by releasing growth factors from fibroblasts and stimulate the healing process. This study was designed to evaluate the influence of laser photostimulation on collagen production in experimentally tenotomized and repaired rabbit Achilles tendons.

Laser photostimulation accelerates wound healing in diabetic rats.

In this study, we examined the hypothesis that laser photostimulation can facilitate healing of impaired wounds in experimental diabetes using a rat model. Diabetes was induced in male rats by streptozotocin injection and two 6 mm diameter circular wounds were created on either side of the spine. The left wound of each animal was treated with a 632.8nm He:Ne laser at a dose of 1.0J/cm2 for five days a week until the wounds closed (three weeks). Measurements of the biomechanical properties of the laser‐treated wounds indicated there was a marginal increase in maximum load (16%), stress (16%), strain (27%), energy absorption (47%) and toughness (84%) compared to control wounds of diabetic rats. Biochemical assays revealed that the amount of total collagen was significantly increased in laser treated wounds (274 ± 8.7 μg) over the control wounds (230 ± 8.4 μg). Sequential extractions of collagen from healing wounds showed that laser treated wounds had significantly greater concentrations of neutral salt soluble (15%) and insoluble collagen (16%) than control wounds, suggesting accelerated collagen production in laser treated wounds. There was an appreciable decrease in pepsin soluble collagen (19%) in laser treated wounds over control wounds, indicating higher resistance to proteolytic digestion. In conclusion, the biomechanical and biochemical results collectively suggest that laser photostimulation promotes the tissue repair process by accelerating collagen production and promoting overall connective tissue stability in healing wounds of diabetic rats.

The biomechanical integrity of bone in experimental diabetes

Patients with diabetes mellitus incur a higher incidence of fractures compared to healthy individuals. This suggests that the structural integrity of the skeletal system may be compromised. To examine the biomechanical consequences of diabetes, we studied the structural integrity of the femur and tibia of rats with streptozotocin-induced diabetes. The induction of diabetes was confirmed by measuring blood glucose levels (>300 mg/dl). Seven-weeks following the establishment of diabetes, the animals were euthanized and the hind limbs removed. The femur and tibia of each hind limb were excised, and prepared for three point bending test on an Instron Materials Testing System. The results revealed a 37% decrease in maximum load (breaking strength) of the femur of diabetic rats when compared to controls. The diabetic femurs had 25% less deformation at maximum load compared to controls. Similarly, energy absorption capacity to yield point and toughness were reduced by 27 and 34%, respectively, in the diabetic femur. A 38% increase in the bending stiffness was observed in the femurs of diabetic rats. Similar results were obtained with the tibias of both groups. Measurement at the break point revealed that the bones of diabetic rats bore significantly less load, deformation and energy absorption capacity than controls. Overall, our findings warrant the conclusion that the diabetic state is associated with mechanical deterioration of bone, resulting in bones with inferior biomechanical integrity.

Nucleoplasmic and cytoplasmic differences in the fluorescence properties of the calcium indicator Fluo-3

The fluorescent indicator Fluo-3 is widely used to monitor the calcium concentration ([Ca2+]) in the cytoplasm and nucleus of various cells. Estimates of nuclear [Ca2+] are based on the assumption of identical behavior of Fluo-3 in different cellular compartments. The assumption is not valid if the fluorescence properties of the dye are altered by the nuclear environment, independent of the [Ca2+]. To determine the effects of the nucleoplasm on the behavior of Fluo-3, we applied laser scanning confocal microscopy and spectrophotometry to measure fluorescence intensity as well as emission and absorbance spectra of the Ca2+ indicator, Fluo-3. Spectra were measured in intact Xenopus oocytes, neuroblastoma cells, and cytoplasmic and nucleoplasmic homogenates. The fluorescence signal in intact cells loaded with Fluo-3 was approximately 2-times higher in the nucleus when compared to the cytoplasm. The fluorescence intensity of Fluo-3 in nucleoplasmic homogenates was higher than in cytoplasmic homogenates or internal buffers even when [Ca2+] was clamped. Despite identical [Ca2+], pH, and temperature, the emission and absorbance spectra of Fluo-3 from nuclear homogenates displayed a higher fluorescence at each wavelength measured when compared to spectra from cytoplasmic homogenates or internal buffer solutions, and saturated above 100 nM. These findings demonstrate that the composition of the nucleoplasm changes the fluorescence properties of the calcium indicator Fluo-3. Consequently, analysis of nuclear calcium dynamics must take into account the distinct behavior of Fluo-3 in different cellular compartments.

Interleukin-6 Production by Endothelial Cells via Stimulation of Protease-Activated Receptors Is Amplified by Endotoxin and Tumor Necrosis Factor-α

Human endothelial cells respond to extracellular proteases, endotoxin (lipopolysaccharide, LPS), and inflammatory cytokines. Endothelial cells express several protease-activated receptors (PAR), including the thrombin-activated receptors PAR-1 and PAR-3 and a thrombin-independent, protease-activated receptor, PAR-2. To examine the potential cooperation between PAR and inflammatory stimuli, we investigated the effects of the PAR-1 agonist peptide Ser-Phe-Leu-Leu-Arg-Asn (SFLLRN) and PAR-2 agonist peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV) on endothelial cells. Human umbilical vein endothelial cells (HUVEC) were cultured in vitro with SFLLRN or SLIGKV in the presence and absence of LPS or tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) levels in the culture supernatants were assayed. Both SFLLRN and SLIGKV induced detectable levels of IL-6 production in a dose-dependent fashion, with the PAR-1 receptor agonist being more potent. In the presence of all stimulatory concentrations of LPS or TNF-alpha tested, both peptides were found to further enhance IL-6 production. The effects of SFLLRN and SLIGKV were specific, as related peptides with identical amino acid compositions, but lacking in consensus sequences, were biologically inactive either alone or in the presence of LPS. Both the direct and the amplifying effects of PAR agonist peptides on IL-6 production were pertussis toxin sensitive and caused an increase in the intracellular levels of calcium, implicating G-proteins and calcium mobilization in these pathways. Furthermore, the amplifying effect of LPS or TNF-alpha on PAR-mediated cytokine production was associated with corresponding increases in nuclear NF-kappaB proteins. The results demonstrate significant potentiation of PAR-induced signaling by LPS and TNF-alpha and indicate the potential cooperation of proteases and inflammatory stimuli in amplifying vascular inflammation.

CXCL10-induced cell death in neurons: role of calcium dysregulation

Chemokines play a key role in the regulation of central nervous system disease. CXCL10 over‐expression has been observed in several neurodegenerative diseases, including multiple sclerosis, Alzheimers disease and HIV‐associated dementia. More recent studies by others and us have shown that CXCL10 elicits apoptosis in fetal neurons. The mechanism of CXCL10‐mediated neurotoxicity, however, remains unclear. In this study, we provide evidence for the direct role of Ca2+ dysregulation in CXCL10‐mediated apoptosis. We demonstrate that treatment of fetal neuronal cultures with exogenous CXCL10 produced elevations in intracellular Ca2+ and that this effect was modulated via the binding of CXCL10 to its cognate receptor, CXCR3. We further explored the association of intracellular Ca2+ elevations with the caspases that are involved in CXC10‐induced neuronal apoptosis. Our data showed that increased Ca2+, which is available for uptake by the mitochondria, is associated with membrane permeabilization and cytochrome c release from this compartment. The released cytochrome c then activates the initiator active caspase‐9. This initiator caspase sequentially activates the effector caspase‐3, ultimately leading to apoptosis. This study identifies the temporal signaling cascade involved in CXCL10‐mediated neuronal apoptosis and provides putative targets for pharmaceutical intervention of neurological disorders associated with CXCL10 up‐regulation.

Involvement of TRPC Channels in CCL2-Mediated Neuroprotection against Tat Toxicity

Chemokine (C-C motif) ligand 2 (CCL2), also known as monocyte chemoattractant protein-1, plays a critical role in leukocyte recruitment and activation. In the present study, we identify an additional role for CCL2 that of neuroprotection against HIV-1 transactivator protein (Tat) toxicity in rat primary midbrain neurons. Furthermore, we report the involvement of transient receptor potential canonical (TRPC) channels in CCL2-mediated neuroprotection. TRPC are Ca2+-permeable, nonselective cation channels with a variety of physiological functions. Blockage of TRPC channels resulted in suppression of both CCL2-mediated neuroprotection and intracellular Ca2+ elevations. Parallel but distinct extracellular signal-regulated kinase (ERK)/cAMP response element-binding protein (CREB) and Akt/nuclear factor κB (NF-κB) pathways were involved in the CCL2-mediated neuroprotection. Blocking TRPC channels and specific downregulation of TRPC channels 1 and 5 resulted in suppression of CCL2-induced ERK/CREB activation but not Akt/NF-κB activation. In vivo relevance of these findings was further corroborated in wild-type and CCR2 knock-out mice. In the wild-type but not CCR2 knock-out mice, exogenous CCL2 exerted neuroprotection against intrastriatal injection of HIV-1 Tat. These findings clearly demonstrate a novel role of TRPC channels in the protection of neurons against Tat through the CCL2/CCR2 axis.

Matrix remodeling in healing rabbit Achilles tendon

Biochemical, biomechanical and ultrastructural properties of the connective tissue matrix were investigated during the early remodeling phase of tissue repair in experimentally tenotomized and repaired rabbit Achilles tendons. Sterile surgical tenotomy was performed on the right Achilles tendons of 14 rabbits and allowed to heal for 15 days. The animals were euthanized and the Achilles tendons excised from both limbs. The left contralateral Achilles tendon of each rabbit was used as a control in the experiments. Prior to biochemical analysis, both intact and healing tendons were tested for their biomechanical integrity. The results revealed that the healing tendons had regained some of their physicochemical characteristics, but differed significantly from the intact left tendons. The healing tendons regained 48% tensile strength, 30% energy absorption, 20% tensile stress, and 14% Young’s modulus of elasticity of intact tendons. In contrast, biochemical analysis showed that the healing tendons had 80% of the collagen and 60% of the collagen crosslinks (hydroxypyridinium) of normal tendons. Sequential extraction of collagen from the tissues yielded more soluble collagen in the healing tendons than intact tendons, suggesting either an increase in collagen synthesis and/or enhanced resorption of mature collagen in healing tendons compared to intact tendons. Electron microscopic studies revealed remarkable differences in the ultrastructure between intact and healing tendons. These observations could explain, in part, the connective tissue response to healing during the early phases of tissue remodeling.

Resistance exercise training lowers HbA1c more than aerobic training in adults with type 2 diabetes

BackgroundThe aim of this study was to compare the effects of 10 weeks of resistance or treadmill exercises on glycemic indices levels prior to and immediately following exercise in adults with type 2 diabetes.Research Design and MethodTwenty inactive subjects (mean age 53.5 years) with type 2 diabetes enrolled in the study. Baseline HbA1c, blood glucose levels, heart rate, and blood pressure were measured for each subject prior to the initiation of the exercise program. Subsequently, subjects were matched to age, waist circumference and sex and assigned to either isocaloric resistance or treadmill exercise groups, which met 3 times per week for 10 weeks.ResultsBoth groups showed a reduction in pre and post-exercise blood glucose and HbA1c values. There was no change in resting blood pressure or heart rate in either group during the course of the 10 week intervention. The group receiving resistance exercises showed significant differences in the daily pre-exercise plasma glucose readings between the beginning and end of the exercise protocol (p < 0.001). There were significant improvements in the mean HbA1c reading pre and post training in both groups (p < 0.001). However, the greater reduction was noted in the resistance exercise group, and at 10 weeks their HbA1c levels were significantly lower than the group that received treadmill exercises (p < 0.006).ConclusionTen weeks of resistance exercises were associated with a significantly better glycemic control in adults with type 2 diabetes compared to treadmill exercise.

Endurance exercise promotes cardiorespiratory rehabilitation without neurorestoration in the chronic mouse model of Parkinsonism with severe neurodegeneration

Physical rehabilitation with endurance exercise for patients with Parkinsons disease has not been well established, although some clinical and laboratory reports suggest that exercise may produce a neuroprotective effect and restore dopaminergic and motor functions. In this study, we used a chronic mouse model of Parkinsonism, which was induced by injecting male C57BL/6 mice with 10 doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (25 mg/kg) and probenecid (250 mg/kg) over 5 weeks. This chronic parkinsonian model displays a severe and persistent loss of nigrostriatal neurons, resulting in robust dopamine depletion and locomotor impairment in mice. Following the induction of Parkinsonism, these mice were able to sustain an exercise training program on a motorized rodent treadmill at a speed of 18 m/min, 0 degrees of inclination, 40 min/day, 5 days/week for 4 weeks. At the end of exercise training, we examined and compared their cardiorespiratory capacity, behavior, and neurochemical changes with that of the probenecid-treated control and sedentary parkinsonian mice. The resting heart rate after 4 weeks of exercise in the chronic parkinsonian mice was significantly lower than the rate before exercise, whereas the resting heart rate at the beginning and 4 weeks afterward in the control or sedentary parkinsonian mice was unchanged. Exercised parkinsonian mice also recovered from elevated electrocardiogram R-wave amplitude that was detected in the parkinsonian mice without exercise for 4 weeks. The values of oxygen consumption, carbon dioxide production, and body heat generation in the exercised parkinsonian mice before and during the Bruce maximal exercise challenge test were all significantly lower than that of their sedentary counterparts. Furthermore, the exercised parkinsonian mice demonstrated a greater mass in the left ventricle of the heart and an increased level of citrate synthase activity in the skeletal muscles. The amphetamine-induced, dopamine release-dependent locomotor activity was markedly inhibited in the sedentary parkinsonian mice and was also inhibited in the exercised parkinsonian mice. Finally, neuronal recovery from the loss of nigrostriatal tyrosine hydroxylase expression and dopamine levels in the severe parkinsonian mice after exercise was not evident. Taken all together, these data suggest that 4 weeks of treadmill exercise promoted physical endurance, resulting in cardiorespiratory and metabolic adaptations in the chronic parkinsonian mice with severe neurodegeneration without demonstrating a restorative potential for the nigrostriatal dopaminergic function.