Experimental study of survival of pedicled perforator flap with flow-through and flow-end blood supply.

BACKGROUND: Flap viability after transfer depends on blood flow from the arterial blood supply below the fascia. This study evaluated survival of a pedicle flap with a perforator lateral branch and flow-through blood supply, compared with that of a flap with a flow-end blood supply and perforator terminal branch. METHODS: Forty Sprague-Dawley rats, 20 in each group, were assigned to transfer of a superficial epigastric artery pedicle island flap with a flow-through or flow-end configuration of blood supply. Laser Doppler imaging was used to evaluate flap perfusion 2 h, 3 days and 5 days after surgery. The rats were killed on day 5, and lead oxide-gelatine-enhanced flap angiography and histology with haematoxylin and eosin staining was performed. Dorsal midline tissue was excised for quantification of vascular endothelial growth factor by western blot assay. RESULTS: On day 5 after surgery, the flow-through group exhibited a significantly greater mean(s.d.) flap survival area (97·8(3·5) versus 80·8(10·2) per cent; P = 0·003), microvascular density (303(19) versus 207(41) per mm(2) ; P < 0·001) and perfusion (8·64(0·14) versus 5·95(0·14) perfusion units; P < 0·001) than the flow-end group. The flow-through group exhibited more angiosomes connected by dilated vascular anastomoses between the skin and subcutaneous fasciae. CONCLUSION: The flow-through blood supply improved pedicle perforator flap survival. Surgical relevance Perforator flap failure is mainly the result of impaired blood supply, as a flow-end blood configuration is nourished only by the perforator terminal branch of the artery. This work showed that the flow-through blood supply nourished by the perforator lateral branch improved flap survival, with dilatation of collateral vascular anastomoses and increased neoangiogenesis. The use of a flow-through configuration improves perforator flap survival and could therefore minimize morbidity resulting from flap necrosis.

Properties of isolated GABAB-mediated inhibitory postsynaptic currents in hippocampal pyramidal cells.

Whole-cell recording techniques were used to record isolated slow inhibitory postsynaptic currents in CA1 pyramidal neurons from rat hippocampal slices. Application of 6-cyano-7-nitroquinoxaline-2,3-dione and 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid eliminated excitatory synaptic transmission, resulting in a 38% reduction in slow inhibitory postsynaptic current magnitude. Subsequent addition of the GABAA antagonist picrotoxin caused a further decrease in slow inhibitory postsynaptic current amplitude. The remaining, isolated slow inhibitory postsynaptic current was blocked by the GABAB antagonist 2-hydroxysaclofen and when cesium was substituted for intracellular potassium. The kinetics of isolated slow inhibitory postsynaptic currents were characterized by single exponential, fourth power activation, and double exponential inactivation. These slow inhibitory postsynaptic currents had a reversal potential of -85.7 +/- 1.6 mV, and a slope conductance of 935 +/- 277 pS. Single slow inhibitory postsynaptic currents carried a total charge flux of 13.4 +/- 7.6 pC. Repetitive stimulation up to 1 Hz progressively reduced steady-state slow inhibitory postsynaptic current amplitude. This attenuation was characterized by a decrease in slope conductance, but slow inhibitory postsynaptic current reversal potential remained unchanged, as did slow inhibitory postsynaptic current kinetics. These results indicate that, under physiological conditions, both ionotropic glutamate- and GABAA-mediated transmission contribute to slow inhibitory postsynaptic current recruitment. Given this finding, activity-dependent decreases in GABAA transmission could contribute to slow inhibitory postsynaptic current depression, though not exclusively, since isolated slow inhibitory postsynaptic currents also demonstrated this property. The use-dependent depression of isolated slow inhibitory postsynaptic currents may be a consequence of a reduction in transmitter release.

Activity-dependent depression of monosynaptic fast IPSCs in hippocampus: contributions from reductions in chloride driving force and conductance.

Whole-cell recordings techniques were used to record pharmacologically isolated fast inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal neurons from rat hippocampal slices. Repetitive extracellular stimulation up to 10 Hz progressively reduced steady-state fast IPSC amplitude. At low stimulation frequencies (up to 1 Hz), this attenuation was characterized by a positive shift of IPSC reversal potential with no change in IPSC conductance. Above 1 Hz stimulation, fast IPSC depression was associated with changes in both reversal potential and IPSC conductance. Use-dependent depression at low frequencies was prevented when cells were chloride-loaded using cesium chloride based intracellular solutions. These findings suggest that activity-dependent depression of fast IPSCs at low stimulus frequencies results entirely from a reduction in chloride driving force, stemming from intracellular chloride accumulation. Activity-dependent changes in fast IPSC conductance occur only at stimulation rates above 1 Hz.

Both survival and development of spontaneously active rat hypothalamic neurons in dissociated culture are dependent on membrane depolarization.

Suppression of endogenous electrical activity was found to have an adverse effect on the survival and bioelectric development of dissociated, embryonic rat hypothalamic neurons in long-term culture. Cultures were treated during the first two weeks in vitro with tetrodotoxin (TTX), a selective blocker of voltage-gated sodium channels, alone and in combination with high extracellular KCl ([K+]o), a membrane depolarizer. Neuron survival was assessed through cell counting experiments, while the development of spontaneous electrical activity was examined with extracellular, patch-electrode recordings. TTX caused both a decrease in cell survival and a decrease in spontaneously active cells; concurrent treatment with K+ protected cells from the adverse effects of TTX. K+ treatment alone increased the fraction of spontaneously active neurons without significantly affecting cell survival. When taken together, these results suggest that the long-term survival of active cells depends on continual membrane depolarization. From these observations, we conclude that there exists two populations of neurons: the electrically active population, whose survival is sensitive to electrical activity, and the quiescent population, whose survival is not.

Development of spontaneous electrical activity by rat hypothalamic neurons in dissociated culture.

The development of spontaneous electrical activity by embryonic rat hypothalamic neurons in dissociated culture was monitored using an extracellular, patch electrode recording method. Embryonic day 17 hypothalamic neurons were plated onto a feeder layer of astrocytes obtained from neonatal rat cerebral cortex, and extracellular electrical activity was monitored beginning the day after plating. The rates and patterns of spontaneous discharge were examined using interpike interval histograms. The percentage of spontaneously active neurons increased steadily with time in culture, from 13% in the first week to 56% during the sixth week in vitro. Although the percentage of spontaneously active cells increased, average firing rates did not change with time in culture. The pattern of electrical discharge was primarily random at all time points, with a small number of cells displaying regular activity while 4 cells were classified as phasic/bursty. In general, spontaneous action potential discharge was not dependent on synaptic transmission, as activity persisted after perfusion with bath solution containing either low Ca2+/high Mg2+ or kynurenic acid. Tetrodotoxin consistently and reversibly abolished spontaneous firing, regardless of culture age. We conclude that spontaneous activity in low density hypothalamic culture develops gradually though steadily, and is generated through an endogenous mechanism, independent of synaptic excitation.

Astrocyte topography and tenascin cytotactin expression: correlation with the ability to support neuritic outgrowth.

We have observed a heterogeneity in the ability of a monolayer of cultured rat astrocytes to support the attachment and growth of dissociated embryonic hypothalamic neurons in culture. Areas of the monolayer which have an uneven surface ('rocky' astrocytes) are poor substrates for neuronal attachment and neuritic outgrowth, while surrounding areas of the glial monolayer ('flat' astrocytes) support extensive neuronal growth. Astrocytes obtained from both neonatal cerebral cortex or hypothalamus displayed 'rocky' morphology. We utilized immunocytochemical techniques with antibodies directed against putative adhesion molecules to investigate the source of this heterogeneity. Antibodies against tenascin/cytotacin, fibronectin, laminin, N-CAM, thrombospondin, heparan sulfate proteoglycan, and the p185 protein product of the neu oncogene were employed in indirect-immunofluorescence experiments. We found that the difference in the surface properties of astrocytes appears to be correlated with the expression of the extracellular matrix molecule tenascin/cytotacin, but not with any of the other molecules we tested. Our data suggest that tenascin/cytotactin is inhibitory to neuronal attachment and process outgrowth in the developing nervous system.

Recruitment of GABAA inhibition in rat neocortex is limited and not NMDA dependent.

1. The recruitment of evoked fast inhibitory postsynaptic currents (IPSCs) and excitatory postsynaptic currents (EPSCs) was examined using whole cell voltage-clamp recordings from layer V pyramidal neurons in slices of rat somatosensory cortex. Synaptic currents were evoked with graded electrical stimulation to assess the relative activation of IPSCs and EPSCs. Fast GABAA ergic IPSCs were selectively recorded by holding cells at potentials equal to EPSC reversal (approximately 0 mV). EPSCs were likewise isolated by holding cells at IPSC reversal potential (about -75 mV). 2. As stimulus intensities were increased, the magnitude of the postsynaptic currents also increased. Over the range of stimuli applied (2-10 V), EPSCs did not exhibit an upper limit. However, fast gamma-aminobutyric acid-A (GABAA-mediated IPSCs reached a maximum at intensities approximately 2 times threshold. 3. The limit on fast inhibition was unresponsive to alterations in N-methyl-D-aspartate (NMDA)-mediated excitation. Exposure to nominally magnesium-free solutions or to the NMDA antagonist 3-[(RS)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid did not affect the fast IPSC maximum. Shifts in the input-output curves for submaximal activation of IPSCs were seen, which were attributed to polysynaptic excitation. 4. Blockade of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate (non-NMDA) receptors with 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) completely abolished synaptically driven, fast GABAA-mediated inhibition. These findings suggested that neocortical inhibitory cells could be driven exclusively through non-NMDA transmission. 5. By comparison, in hippocampal CA1 pyramidal neurons maximal fast inhibition was sensitive to both NMDA and non-NMDA receptor blockade. 6. The results in neocortex were corroborated by direct intracellular recordings from layer V-VI interneurons. Non-NMDA receptor blockade with CNQX prevented synaptic activation of action potentials in these cells, even during cotreatment with magnesium-free solution. 7. Together, these results suggest that recruitment of GABA(A) ergic IPSCs in neocortex is ultimately driven via glutamatergic afferents arriving at non-NMDA receptors on interneurons. Properties limiting fast inhibition would favor the propagation of enhanced excitatory activity through the neuronal network.

Restrictions on inhibitory circuits contribute to limited recruitment of fast inhibition in rat neocortical pyramidal cells.

To further define the operational boundaries on fast inhibition in neocortex, whole cell recordings were made from layer V pyramidal neurons in neocortical slices to evaluate evoked inhibitory postsynaptic currents (IPSCs) and spontaneous miniature IPSCs (mIPSCs). Stimulating electrodes were placed in layers VI and I/II to determine whether simultaneous stimulation of deep and superficial laminae could extend the magnitude of maximal IPSCs evoked by deep-layer stimulation alone. The addition of superficial-layer stimulation did not increase maximal IPSC amplitude, confirming the strict limit on fast inhibition. Spontaneous miniature IPSCs were recorded in the presence of tetrodotoxin. The frequency of spontaneous mIPSCs ranged from 10.0 to 33.1 Hz. mIPSC amplitude varied considerably, with a range of 5. 0-128.2 pA and a mean value of 20.7+/-4.1 pA (n = 12 cells). The decay phase of miniature IPSCs was best fit by a single exponential, similar to evoked IPSCs. The mean time constant of decay was 6.4+/-0.6 ms, with a range of 0.2-20.1 ms. The mean 10-90% rise time was 1.9+/-0.2 ms, ranging from 0.2 to 6.3 ms. Evaluation of mIPSC kinetics revealed no evidence of dendritic filtering. Amplitude histograms of mIPSCs exhibited skewed distributions with several discernable peaks that, when fit with Gaussian curves, appeared to be spaced equidistantly, suggesting that mIPSC amplitudes varied quantally. The mean separation of Gaussian peaks ranged from 6.1 to 7.8 pA. The quantal distributions did not appear to be artifacts of noise. Exposure to saline containing low Ca(2+) and high Mg(2+) concentrations reduced the number of histogram peaks, but did not affect the quantal size. Mean mIPSC amplitude and quantal size varied with cell holding potential in a near-linear manner. Statistical evaluation of amplitude histograms verified the multimodality of mIPSC amplitude distributions and corroborated the equidistant spacing of peaks. Comparison of mIPSC values with published data from single GABA channel recordings suggests that the mean mIPSC conductance corresponds to the activation of 10-20 GABA(A) receptor channels, and that the release of a single inhibitory quantum opens 3-6 channels. Further comparison of mIPSCs with evoked inhibitory events suggests that a single interneuron may form, on average, 4-12 functional synapses with a pyramidal cell, and that 10-12 individual interneurons are engaged during recruitment of maximal population IPSCs. This suggests that inhibitory circuits are much more restricted in both the size of the unit events and effective number of connections when compared with excitatory inputs.

Synchronous firing of inhibitory interneurons results in saturation of fast GABA(A) IPSC magnitude but not saturation of fast inhibitory efficacy in rat neocortical pyramidal cells.

The kinetic properties of evoked fast inhibitory postsynaptic currents were examined to elucidate factors underlying the limit on the magnitude of fast inhibition in neocortex. Using whole-cell voltage-clamp recordings from layer V pyramidal neurons in slices of rat somatosensory cortex, fast gamma-aminobutyric acid-A (GABA[A])ergic inhibitory postsynaptic currents were selectively recorded by holding cells at potentials equal to excitatory postsynaptic current reversal (approximately 0 mV). As stimulus intensity was increased, the magnitude and duration of the fast inhibitory postsynaptic current increased. Over the range of stimuli applied (2-10 V), fast GABA(A)-mediated inhibitory postsynaptic currents reached a maximum peak conductance of 25.9 +/- 4.2 nS (range 10.5-41.2 nS) at intensities approximately 2-times threshold. As stimulus intensities were increased beyond this point of maximal conductance, the time constant of current decay increased as function of stimulus strength, while rise time remained unaffected. Exposure to nominally magnesium-free solutions did not affect maximal peak conductances of fast inhibitory postsynaptic currents, but did cause an increase in the time constants of current decay by 66.3 +/- 23.6%, resulting in an 85.6 +/- 24.6% increase in the total charge flux carried by single inhibitory postsynaptic currents. This effect may be due to prolonged activation of postsynaptic GABA(A) receptors by excess GABA released in response to increased excitation. Exposure to the GABA uptake blocker, nipecotic acid, similarly prolonged current decay without affecting the maximal peak conductance. Our findings suggest that the limit on recruitment of evoked fast inhibition in neocortical layer V pyramidal cells arises from the saturation of postsynaptic GABA(A) receptors. However, while there is a limit to the peak fast inhibitory postsynaptic conductance which can be recruited with increasing excitation, inhibitory strength may still be modulated by increasing charge flux through the prolongation of fast inhibition.