T.P. Tran

Tourniquet-induced acute ischemia-reperfusion injury in mouse skeletal muscles: Involvement of superoxide

Although arterial limb tourniquet is one of the first-line treatments to prevent exsanguinating hemorrhage in both civilian pre-hospital and battlefield casualty care, prolonged application of a limb tourniquet can lead to serious ischemia-reperfusion injury. However, the underlying pathomechanisms of tourniquet-induced ischemia-reperfusion injury are still poorly understood. Using a murine model of acute limb ischemia-reperfusion, we investigated if acute limb ischemia-reperfusion injury is mediated by superoxide overproduction and mitochondrial dysfunction. Hind limbs of C57/BL6 mice were subjected to 3h ischemia and 4h reperfusion via placement and release of a rubber tourniquet at the greater trochanter. Approximately 40% of the gastrocnemius muscle suffered infarction in this model. Activities of mitochondrial electron transport chain complexes including complex I, II, III, and IV in the gastrocnemius muscle were decreased in the ischemia-reperfusion group compared to sham. Superoxide production was increased while activity of manganese superoxide dismutase (MnSOD, the mitochondria-targeted SOD isoform) was decreased in the ischemia-reperfusion group compared to the sham group. Pretreatment with tempol (a SOD mimetic, 50mg/kg) or co-enzyme Q(10) (50mg/kg) not only decreased the superoxide production, but also reduced the infarct size and normalized mitochondrial dysfunction in the gastrocnemius muscle. Our results suggest that tourniquet-induced skeletal muscle ischemia-reperfusion injuries including infarct size and mitochondrial dysfunction may be mediated via superoxide overproduction and reduced antioxidant activity. In the future, this murine ischemia-reperfusion model can be adapted to mechanistically evaluate anti-ischemic molecules in tourniquet-induced skeletal muscle injury.

Blunted excitability of aortic baroreceptor neurons in diabetic rats: involvement of hyperpolarization-activated channel.

AIMS Although dysfunction of arterial baroreflex occurs in human and animal models of type-1 diabetes (T1D), the mechanisms involved in the impairment of the baroreflex still remain unclear. The nodose ganglion (NG) contains the cell bodies of the aortic baroreceptor (AB) neurons. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed in AB neurons and play an important role in regulating the cell excitability. We investigated whether the excitability of AB neurons is depressed in streptozotocin (STZ)-induced T1D rats and whether HCN channels are involved in this depression. METHODS AND RESULTS Using the whole-cell patch clamp technique, we found that AB neuron excitability (action potential frequency at 50 pA current stimulation) in the T1D rats was lower than that in the sham rats (0.4 +/- 0.5 vs. 4.8 +/- 0.6 spikes/s, P < 0.05; AB neurons were identified by DiI staining). In addition, HCN current density in AB neurons from the T1D rats was bigger than that from the sham rats (60.2 +/- 6.1 vs. 30.7 +/- 4.9 pA/pF at test pulse -140 from holding potential -40 mV, P < 0.05). Furthermore, HCN channel blockers (5 mM cesium chloride and 100 microM ZD7288) significantly reduced HCN currents and increased action potential frequency of the AB neurons in sham and T1D rats. Immunofluorescent and western blot analyses demonstrated that the expression of HCN1 and HCN2 channel protein in the NG from the T1D rats was higher than that from the sham rats. CONCLUSION These results indicate that the HCN channels influence the excitability of AB neurons, and more importantly, contribute to the decreased excitability of AB neurons in T1D rats.

Diabetes alters protein expression of hyperpolarization-activated cyclic nucleotide-gated channel subunits in rat nodose ganglion cells.

Vagal afferent neurons, serving as the primary afferent limb of the parasympathetic reflex, could be involved in diabetic autonomic neuropathy. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed in the vagal afferent neurons and play an important role in determining cell membrane excitation. In the present study, the protein expression and the electrophysiological characteristics of HCN channels were investigated in nodose ganglion (NG) afferent neurons (A-fiber and C-fiber neurons) from sham and streptozotocin (STZ)-induced diabetic rats. In the sham NG, HCN1, HCN3, and HCN4 were expressed in the A-fiber neurons; and HCN2, HCN3, and HCN4 were expressed in the C-fiber neurons. Compared to the sham NG neurons, diabetes induced the expression of HCN2 in the A-fiber neurons besides overexpression of HCN1 and HCN3; and enhanced the expression of HCN2 and HCN3 in C-fiber neurons. In addition, whole-cell patch-clamp data revealed diabetes also increased HCN currents in A-fiber and C-fiber neurons. However, we found that diabetes did not alter the total nodose afferent neuron number and the ratio of A-fiber/C-fiber neurons. These results indicate that diabetes induces the overexpression of HCN channels and the electrophysiological changes of HCN currents in the A- and C-fiber nodose neurons, which might contribute to the diabetes-induced alteration of cell excitability in the vagal afferent neurons.

Reduced expression and activation of voltage-gated sodium channels contributes to blunted baroreflex sensitivity in heart failure rats†

Voltage‐gated sodium (Nav) channels are responsible for initiation and propagation of action potential in the neurons. To explore the mechanisms of chronic heart failure (CHF)‐induced baroreflex dysfunction, we measured the expression and current density of Nav channel subunits (Nav1.7, Nav1.8, and Nav1.9) in the aortic baroreceptor neurons and investigated the role of Nav channels in aortic baroreceptor neuron excitability and baroreflex sensitivity in sham and CHF rats. CHF was induced by left coronary artery ligation. The development of CHF (6–8 weeks after the coronary ligation) was confirmed by hemodynamic and morphological characteristics. Immunofluorescent data indicated that Nav1.7 was expressed in A‐type (myelinated) and C‐type (unmyelinated) nodose neurons, but Nav1.8 and Nav1.9 were expressed only in C‐type nodose neurons. Real‐time RT‐PCR and Western blot data showed that CHF reduced mRNA and protein expression levels of Nav channels in nodose neurons. In addition, using the whole‐cell patch‐clamp technique, we found that Nav current density and cell excitability of the aortic baroreceptor neurons were lower in CHF rats than that in sham rats. Aortic baroreflex sensitivity was blunted in anesthetized CHF rats, compared with that in sham rats. Furthermore, Nav channel activator (rATX II, 100 nM) significantly enhanced Nav current density and cell excitability of aortic baroreceptor neurons and improved aortic baroreflex sensitivity in CHF rats. These results suggest that reduced expression and activation of the Nav channels are involved in the attenuation of baroreceptor neuron excitability, which subsequently contributes to the impairment of baroreflex in CHF state.

Mitochondria-Derived Superoxide Links to Tourniquet-Induced Apoptosis in Mouse Skeletal Muscle

Our previous study has reported that superoxide mediates ischemia-reperfusion (IR)-induced necrosis in mouse skeletal muscle. However, it remains poorly understood whether IR induces apoptosis and what factors are involved in IR-induced apoptosis in skeletal muscle. Using a murine model of tourniquet-induced hindlimb IR, we investigated the relationship between mitochondrial dysfunction and apoptosis in skeletal muscle. Hindlimbs of C57/BL6 mice were subjected to 3 h ischemia and 4 h reperfusion via placement and release of a rubber tourniquet at the greater trochanter. Compared to sham treatment, tourniquet-induced IR significantly elevated mitochondria-derived superoxide production, activated opening of mitochondrial permeability transition pore (mPTP), and caused apoptosis in the gastrocnemius muscles. Pretreatment with a superoxide dismutase mimetic (tempol, 50 mg/kg) or a mitochondrial antioxidant (co-enzyme Q10, 50 mg/kg) not only decreased mitochondria-derived superoxide production, but also inhibited mPTP opening and apoptosis in the IR gastrocnemius muscles. Additionally, an inhibitor of mPTP (cyclosporine A, 50 mg/kg) also inhibited both mPTP opening and apoptosis in the IR gastrocnemius muscles. These results suggest that mitochondria-derived superoxide overproduction triggers the mPTP opening and subsequently causes apoptosis in tourniquet-induced hindlimb IR.

Alterations of calcium channels and cell excitability in intracardiac ganglion neurons from type 2 diabetic rats

Clinical study has demonstrated that patients with type 2 diabetes with attenuated arterial baroreflex have higher mortality rate compared with those without arterial baroreflex dysfunction. As a final pathway for the neural control of the cardiac function, functional changes of intracardiac ganglion (ICG) neurons might be involved in the attenuated arterial baroreflex in the type 2 diabetes mellitus (T2DM). Therefore, we measured the ICG neuron excitability and Ca(2+) channels in the sham and T2DM rats. T2DM was induced by a combination of both high-fat diet and low-dose streptozotocin (STZ, 30 mg/kg ip) injection. After 12-14 wk of the above treatment, the T2DM rats presented hyperglycemia, hyperlipidemia, and insulin resistance but no hyperinsulinemia, which closely mimicked the clinical features of the patients with T2DM. Data from immunofluorescence staining showed that L, N, P/Q, and R types of Ca(2+) channels were expressed in the ICG neurons, but only protein expression of N-type Ca(2+) channels was decreased in the ICG neurons from T2DM rats. Using whole cell patch-clamp technique, we found that T2DM significantly reduced the Ca(2+) currents and cell excitability in the ICG neurons. ω-Conotoxin GVIA (a specific N-type Ca(2+) channel blocker, 1 μM) lowered the Ca(2+) currents and cell excitability toward the same level in sham and T2DM rats. These results indicate that the decreased N-type Ca(2+) channels contribute to the suppressed ICG neuron excitability in T2DM rats. From this study, we think high-fat diet/STZ injection-induced T2DM might be an appropriate animal model to test the cellular and molecular mechanisms of cardiovascular autonomic dysfunction.