Intracortical excitation of spiny neurons in layer 4 of cat striate cortex in vitro

Recordings were made from pairs of neurons in cat striate visual cortex in vitro to study the AMPA-channel-mediated components of intracortical excitatory synaptic connections between layer 4 spiny neurons and between layer 6 and layer 4 spiny neurons. Forty-six of the 72 cells recorded were identified morphologically. They consisted of spiny stellate and pyramidal cells in layer 4, and pyramidal cells in layer 6. Connections between layer 4 excitatory cells involve excitatory postsynaptic potentials (EPSPs) averaging 949 microV, with an average coefficient of variation of 0.21 (n = 30). The synapses operate at very high release probabilities (0.69-0.98). With repetitive stimulation these EPSPs show varying degrees of depression, largely mediated by presynaptic changes in release probability. Four pairs of layer 4 cells were reciprocally connected. The connections from layer 6 to layer 4 involve smaller, more variable EPSPs, with an average amplitude of 214 microV, and average coefficient of variation 0.72 (n = 7). These synapses operate at moderately high release probabilities (0.37-0.56). They show facilitation with repetitive stimulation, mediated largely by presynaptic changes in release probability. One excitatory connection from a layer 4 neuron to a layer 6 pyramidal cell was also detected. Thus, layer 4 spiny neurons receive effective excitation from two intracortical sources that have different synaptic dynamics and are likely to contribute significantly to the temporal properties of these cells in vivo.     

Presynaptic frequency filtering in the gamma frequency band; dual intracellular recordings in slices of adult rat and cat neocortex

Using dual intracellular recordings in slices of adult rat and cat neocortex, the frequency-filtering characteristics of 'depressing' synapses made by pyramidal axons at interspike intervals between 5 and 50 ms were studied. At 'depressing' connections from excitatory cells to some inhibitory interneurons (n = 6), recovery from short interspike interval depression was near exponential. Extrapolation of exponentials fitted to this recovery demonstrated a residual 10-20% depression at intervals >50 ms. This slowly decaying component was larger for later excitatory postsynaptic potentials (EPSPs) in trains which were typically more strongly depressed. At >80% of connections between spiny excitatory cells and at pyramid to parvalbumin-immunopositive interneuron connections, however, recovery exhibited a more complex time course. A narrow 'notch' (half-width 5 ms), peaking at intervals of 13-25 ms during which the EPSP was depressed further, interrupted recovery from short interval depression. This 'notch' was equally apparent for all EPSPs in brief trains and was mediated presynaptically.     

Dynamic properties of excitatory synaptic connections involving layer 4 pyramidal cells in adult rat and cat neocortex

To investigate the properties of excitatory connections between layer 4 pyramidal cells and whether these differed between rat and cat, paired intracellular recordings were made with biocytin filling in slices of adult neocortex. These connections were also compared with those from layer 4 spiny cells to layer 3 pyramids and connections between layer 3 pyramids. Connectivity ratios for layer 4 pyramid-pyramid pairs (1:14 cat, 1:18 rat) appeared lower than for the other types of connections studied in parallel, but excitatory postsynaptic potential (EPSP) amplitudes and time course were not significantly different either between species or across types of connection. Layer 4 pyramids targeted postsynaptic basal dendrites in both species, whether the pyramidal target was in layer 4 or layer 3. Within layer 4, relationships between mean EPSP amplitude, numbers of putative contacts, and distance between connected pairs indicated a rapid decline in connectivity strength with distance, equivalent to 3.4 mV and 10 synapses per 100 microm separation, from a maximum of 4 mV and 10 synapses at 0 microm. However, a subset, of burst-firing layer 4 pyramids, appeared to make no connections with other layer 4 spiny cells. Second EPSPs were depressed by 36% in rat and 28% in cat relative to first EPSPs at interspike intervals <15 ms. Subsequent EPSPs in brief trains were further depressed. Depression was predominantly presynaptic in origin. Recovery from depression could not be described adequately by a simple exponential for individual connections; it included peaks and troughs with periodicities of 10-15 ms. Complex relationships between the first 2 interspike intervals and third EPSP amplitude were also apparent in all connections so studied. Large third EPSPs followed specific combinations of first and second interspike intervals so that increasing, or decreasing, one without changing the other resulted in a smaller third EPSP. Finally, the outputs of layer 4 spiny cells to layer 3 exhibited partial recovery from depression during longer high-frequency trains, a property not apparent in the other connections studied.     

Effects of isoflurane and xenon on Ba2+-currents mediated by N-type calcium channels

Background. Isoflurane and xenon are inhalation general anaestheticswith differing clinical profiles and contrasting synaptic actions.Both agents have been shown to depress excitatory synaptic responses.Whether this is via pre-synaptic or post-synaptic mechanismshas not been determined clearly. N-type calcium channels area putative pre-synaptic target for these agents. We tested whetherN-type calcium channels were sensitive to isoflurane and xenonand whether there was any stereoselectivity in the effect ofisoflurane. Methods. We used patch-clamp electrophysiology on isolated HEK293cells stably expressing N-type calcium channels to investigatethe effects of isoflurane and xenon on barium currents mediatedby N-type calcium channels. Results. Racemic isoflurane caused a concentration-dependentreduction (11–35%) in the peak current through the N-typechannels in the concentration range 0.15–1.22 mM. In theclinically relevant concentration range the inhibition was small.At an isoflurane concentration of 0.31 mM (equivalent to 1 MAC),the peak N-type current was inhibited by 14 (1)%. The opticalisomers of isoflurane were found to be equally potent at inhibitingcurrents through N-type channels. The inert gas anaestheticxenon was found to have no measureable effect on N-type channelsat a concentration of 3.4 mM (1 MAC). Conclusions. These results suggest that N-type calcium channelsare not the targets mediating general anaesthesia with thesetwo inhalation agents. Declaration of interest. Professor Franks is a board memberof an Imperial College spin-out company, Protexeon Ltd, thatis interested in developing clinical applications for medicalgases, including xenon. Professor Franks is a paid consultantin this activity. In addition Air Products have funded workin the authors' laboratories that bears on the actions of xenonas an anaesthetic and neuroprotectant. Air Products has a financialstake in Protexeon Ltd.     

Excitatory Synaptic Transmission Mediated by NMDA Receptors Is More Sensitive to Isoflurane than Are Non-NMDA Receptor-mediated Responses

Background: Effects of volatile anesthetic agents on N-methyl-D-aspartate (NMDA) receptor-mediated excitatory synaptic transmission have not been well characterized. The authors compared effects produced by halothane and isoflurane on electrophysiologic properties of NMDA and non-NMDA receptor-mediated synaptic responses in slices from the rat hippocampus.

Methods: Field excitatory postsynaptic potentials (fEPSPs) in the CA1 area were recorded with extracellular electrodes after electrical stimulation of Schaffer-collateral-commissural fiber inputs. NMDA or non-NMDA receptor-mediated fEPSPs were pharmacologically isolated using selective antagonists. Clinically relevant concentrations of halothane or isoflurane were applied to slices in an artificial cerebrospinal fluid perfusate. Paired pulse facilitation was used as a measure of presynaptic effects of the anesthetic agents.

Results: Clinically relevant concentrations of halothane (1.2 vol% [almost equal to] 0.35 mM) depressed fEPSP amplitudes mediated by NMDA receptors and non-NMDA receptors to a similar degree (mean +/- SD: 63.3 +/- 14.0% of control, n = 5; 60.2 +/- 7.3% of control, n = 7, respectively). In contrast, isoflurane (1.4 vol% [almost equal to] 0.50 mM) preferentially depressed fEPSP amplitudes mediated by NMDA receptors (44.0 +/- 7.4% of control, n = 6, P < 0.001) compared with those for non-NMDA receptors (68.7 +/- 5.4% of control, n = 6), indicating a selective, additional postsynaptic effect. Paired pulse facilitation of fEPSPs was increased significantly by both anesthetic agents from 1.37 +/- 0.13 to 1.91 +/- 0.25 (n = 5, P < 0.05 for halothane) and from 1.44 +/- 0.04 to 1.64 +/- 0.08 (n = 5, P < 0.01 for isoflurane), suggesting that presynaptic mechanisms are also involved in fEPSP depression produced by the anesthetic agents. Neither rise times nor decay times of fEPSPs were changed in the presence of the anesthetic agents.     

Layer 6 cortico-thalamic pyramidal cells preferentially innervate interneurons and generate facilitating EPSPs

The properties of the connections made by the axons of pyramidal cells with cortico-thalamic (CT)-like morphology with a range of postsynaptic layer 6 targets were studied with dual intracellular recordings in slices of adult rat and cat neocortex. The cells were filled with biocytin and identified morphologically and, where appropriate, immunofluorescently. CT-like pyramids contacted interneurons with a very high probability (up to 1:2) but contacted other layer 6 pyramidal cells only rarely (approximately 1:80). The excitatory postsynaptic potentials (EPSPs) that they elicited both in pyramidal cells and in a variety of types of interneurons (including those immunopositive for parvalbumin and for somatostatin) facilitated, the second EPSP being larger than the first over a range of interspike intervals. Facilitation was not, however, maximal at the shortest intervals; in fact, depression was apparent at some connections at short interspike intervals. Facilitation in the majority of connections peaked at intervals of 25-35 ms and then declined slowly. Nor did these connections display the augmentation typical of many other strongly facilitating connections. Third EPSPs were smaller on average than second EPSPs, and fourth and subsequent EPSPs could be depressed (relative to first EPSPs). The properties of the outputs of these CT-like pyramidal cells are therefore quite distinct from those of other pyramidal cells, both within layer 6 and in other layers, possibly reflecting their unique role as both first order thalamo-cortical recipient and cortico-thalamic output neurons.     

Fentanyl decreases Ca2+ currents in a population of capsaicin-responsive sensory neurons

BACKGROUND: Neuraxial opioids produce analgesia in part by decreasing excitatory neurotransmitter release from primary nociceptive neurons, an effect that may be due to inhibition of presynaptic voltage-activated Ca2+ channels. The purpose of this study was to determine whether opioids decrease Ca2+ currents (I Ca ) in primary nociceptive neurons, identified by their response to the algogenic agent capsaicin. METHODS: I was recorded from acutely isolated rat dorsal root ganglion neurons using the whole cell patch clamp technique before, during, and after application of the micro -opioid agonist fentanyl (0.01-1 micro m). Capsaicin was applied to each cell at the end of the experiment. RESULTS: Fentanyl reduced I Ca in a greater proportion of capsaicin-responsive cells (62 of 106, 58%) than capsaicin-unresponsive cells (2 of 15, 13%; P < 0.05). Among capsaicin-responsive cells, the decrease in I Ca was 38 +/- 3% (n = 36, 1 micro m) in fentanyl-sensitive cells just 7 +/- 1% (n = 15, 1 micro m; P < 0.05) in fentanyl-insensitive cells. Among capsaicin-responsive cells, I Ca inactivated more rapidly in fentanyl-sensitive cells (tau, 52 +/- 4 ms, n = 22) than in fentanyl-insensitive cells (93 +/- 14 ms, n = 24; P < 0.05). This was not due to differences in the types of Ca2+ channels expressed as the magnitudes of omega-conotoxin GVIA-sensitive (N-type), nifedipine-sensitive (L-type), and GVIA/nifedipine-resistant (primarily P-/Q-type) components of I Ca were similar. CONCLUSIONS: The results show that opioid-sensitive Ca2+ channels are expressed by very few capsaicin-unresponsive neurons but by more than half of capsaicin-responsive neurons. The identity of the remaining capsaicin-responsive (and therefore presumed nociceptive) neurons that express opioid-insensitive Ca2+ channels is unknown but may represent a potential target of future non-opioid-based therapies for acute pain.     

Synaptic efficacy during repetitive activation of excitatory inputs in primate dorsolateral prefrontal cortex

Neurons in the monkey dorsolateral prefrontal cortex (DLPFC) fire persistently during the delay period of working memory tasks. To determine how repetitive firing affects the efficacy of synaptic inputs to DLPFC layer 3 neurons, we examined the effects of repetitive presynaptic stimulation on the amplitude and temporal summation of EPSPs. Recordings were obtained in monkey DLPFC brain slices from regular spiking (RS) pyramidal cells and two types of interneurons, fast spiking (FS) and adapting non-pyramidal (ANP) cells. Repetitive stimulation of presynaptic axons in layer 3 caused EPSP depression in RS and FS neurons, but EPSP facilitation in ANP cells. A shorter EPSP duration produced weaker temporal summation in FS neurons compared to the other cell classes. Thus, due to the combined effects of dynamic changes in EPSP amplitude and differences in temporal summation, the effect of a presynaptic spike train differed according to the postsynaptic cell class. Similar results were obtained when recording unitary EPSPs evoked in connected pairs of presynaptic RS pyramidal cells and postsynaptic RS, FS or ANP neurons. In addition, similar differences in the efficacy of sustained inputs among cell classes were observed when delay-related firing was reproduced in vitro by stimulating inputs with the timing of spike trains recorded from the DLPFC of monkeys performing a delayed-response task. We suggest that the transition from baseline firing rates to higher frequency delay-related firing may lead to the differential activation of distinct cell populations, with corresponding significant effects on the patterns of activity in local prefrontal circuits.     

Fentanyl Decreases Ca2+ Currents in a Population of Capsaicin-responsive Sensory Neurons

Background: Neuraxial opioids produce analgesia in part by decreasing excitatory neurotransmitter release from primary nociceptive neurons, an effect that may be due to inhibition of presynaptic voltage-activated Ca2+ channels. The purpose of this study was to determine whether opioids decrease Ca2+ currents (ICa) in primary nociceptive neurons, identified by their response to the algogenic agent capsaicin.

Methods: ICa was recorded from acutely isolated rat dorsal root ganglion neurons using the whole cell patch clamp technique before, during, and after application of the [mu]-opioid agonist fentanyl (0.01-1 [mu]m). Capsaicin was applied to each cell at the end of the experiment.

Results: Fentanyl reduced ICa in a greater proportion of capsaicin-responsive cells (62 of 106, 58%) than capsaicin-unresponsive cells (2 of 15, 13%;P < 0.05). Among capsaicin-responsive cells, the decrease in ICa was 38 +/- 3% (n = 36, 1 [mu]m) in fentanyl-sensitive cells versus just 7 +/- 1% (n = 15, 1 [mu]m;P < 0.05) in fentanyl-insensitive cells. Among capsaicin-responsive cells, ICa inactivated more rapidly in fentanyl-sensitive cells ([tau]h, 52 +/- 4 ms, n = 22) than in fentanyl-insensitive cells (93 +/- 14 ms, n = 24;P < 0.05). This was not due to differences in the types of Ca2+ channels expressed as the magnitudes of [omega]-conotoxin GVIA-sensitive (N-type), nifedipine-sensitive (L-type), and GVIA/nifedipine-resistant (primarily P-/Q-type) components of ICa were similar.     

Volatile Anesthetics Depress Glutamate Transmission Via Presynaptic Actions

Background: Recent evidence for a presynaptic depression of glutamate release produced by volatile anesthetics prompted the current study of isoflurane and halothane effects on glutamate-mediated transmission in the mammalian central nervous system.

Methods: Electrophysiologic recordings from CA1 neurons in rat hippocampal brain slices were used to measure anesthetic effects on glutamate-mediated excitatory postsynaptic potential (EPSP) amplitudes and paired pulse facilitation. Paired pulse facilitation is known to be altered when the calcium-dependent release of glutamate is depressed, but not when EPSP amplitudes are depressed by postsynaptic mechanisms.

Results: Isoflurane depressed EPSP amplitudes over a concentration range of 0.35-2.8 vol %, with a 50% depression (EC50) occurring at 1.0 vol % (0.71 rat minimum alveolar concentration). This depression was accompanied by an increase in paired-pulse facilitation of approximately 30% at 1.7 vol %, using interpulse intervals of 120 ms. Halothane depressed EPSP amplitudes in a concentration-dependent manner (0.3-2.4 vol %, EC50 = 1.1 minimum alveolar concentration; 1.3 vol %) and also increased facilitation by approximately 20% at 1.2 vol %. These effects persisted in the presence of 10 micro Meter bicuculline, indicating that enhanced gamma-aminobutyric acid-mediated inhibition was not involved. The anesthetic-induced increase in facilitation and EPSP depression was mimicked by lowering extracellular calcium, which is known to depress glutamate release at these synapses. The postsynaptic glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione depressed EPSP amplitudes with no change in facilitation.     

The block of total and N-type calcium conductance in mouse sensory neurons by the local anesthetic n-butyl-p-aminobenzoate

To contribute to the understanding of the mechanism underlying selective analgesia by epidural application of suspensions of the local anesthetic butamben (n-butyl-p-aminobenzoate; BAB), we investigated the effect of dissolved BAB on calcium channels in sensory neurons. Small-diameter dorsal root ganglion neurons from newborn mice were used to measure whole-cell barium or calcium currents through calcium channels upon voltage-clamp stimulation. BAB suppressed the voltage-step-evoked barium current of these cells in a concentration-dependent manner with a 50% inhibitory concentration of 207 +/- 14 microM (n = 40). A similar concentration dependency was found for the pharmacologically isolated N-type component of the whole-cell barium current. The time constants of inactivation and deactivation of the N-type current became smaller in the presence of BAB, thus suggesting that kinetic changes are involved in the inhibition of this current. BAB caused a similar inhibition of the total calcium current and its N-type component when these currents were evoked by command potentials with the shape of an action potential. This inhibition of calcium currents by BAB should be considered in the search for the mechanism of selective analgesia by epidural suspensions of this local anesthetic.     

Volatile Anesthetics Reduce Low-voltage-activated Calcium Currents in a Thyroid C-Cell Line

Background: Volatile anesthetics may act in part by inhibiting voltage-dependent calcium channels. The effects of several volatile agents on three types of calcium channels in a thyroid C-cell line were examined.

Methods: Whole-cell calcium currents were recorded using standard patch clamp techniques. Current-voltage relationships were derived before, during, and after application of isoflurane, enflurane, or halothane. Low-voltage-activated (LVA; T type) calcium currents were isolated based on the voltage range of activation. High-voltage-activated (HVA) calcium currents were separated into L and N types using omega-conotoxin GVIA (omega-CTX) and nicardipine.

Results: All three agents reversibly decreased both LVA and HVA currents at clinically relevant concentrations. Isoflurane and enflurane both reduced peak LVA current more than peak HVA current: -33 +/- 6% (mean +/- SE) versus -22 +/- 4% for 0.71 mM isoflurane (n = 6), and -46 +/- 6% versus -35 +/- 5% for 1.21 mM enflurane (n = 6). In contrast, halothane depressed LVA and HVA currents to a similar extent: -22 +/- 4% versus -29 +/- 3% for 0.65 mM halothane (n = 6). Isoflurane had no effect on LVA whole-cell current kinetics. Pretreatment with either omega-CTX (400 nM) or nicardipine (1 micro Meter) did not change the sensitivity of HVA current to isoflurane.     

Development of inhibitory timescales in auditory cortex

The time course of inhibition plays an important role in cortical sensitivity, tuning, and temporal response properties. We investigated the development of L2/3 inhibitory circuitry between fast-spiking (FS) interneurons and pyramidal cells (PCs) in auditory thalamocortical slices from mice between postnatal day 10 (P10) and P29. We found that the maturation of the intrinsic and synaptic properties of both FS cells and their connected PCs influence the timescales of inhibition. FS cell firing rates increased with age owing to decreased membrane time constants, shorter afterhyperpolarizations, and narrower action potentials. Between FS-PC pairs, excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) changed with age. The latencies, rise, and peak times of the IPSPs, as well as the decay constants of both EPSPs and IPSPs decreased between P10 and P29. In addition, decreases in short-term depression at excitatory PC-FS synapses resulted in more sustained synaptic responses during repetitive stimulation. Finally, we show that during early development, the temporal properties that influence the recruitment of inhibition lag those of excitation. Taken together, our results suggest that the changes in the timescales of inhibitory recruitment coincide with the development of the tuning and temporal response properties of auditory cortical networks.     

Excitatory synaptic transmission mediated by NMDA receptors is more sensitive to isoflurane than are non-NMDA receptor-mediated responses

BACKGROUND: Effects of volatile anesthetic agents on N-methyl-D-aspartate (NMDA) receptor-mediated excitatory synaptic transmission have not been well characterized. The authors compared effects produced by halothane and isoflurane on electrophysiologic properties of NMDA and non-NMDA receptor-mediated synaptic responses in slices from the rat hippocampus. METHODS: Field excitatory postsynaptic potentials (fEPSPs) in the CA1 area were recorded with extracellular electrodes after electrical stimulation of Schaffer-collateral-commissural fiber inputs. NMDA or non-NMDA receptor-mediated fEPSPs were pharmacologically isolated using selective antagonists. Clinically relevant concentrations of halothane or isoflurane were applied to slices in an artificial cerebrospinal fluid perfusate. Paired pulse facilitation was used as a measure of presynaptic effects of the anesthetic agents. RESULTS: Clinically relevant concentrations of halothane (1.2 vol% approximately 0.35 mM) depressed fEPSP amplitudes mediated by NMDA receptors and non-NMDA receptors to a similar degree (mean +/- SD: 63.3 +/- 14.0% of control, n = 5; 60.2 +/- 7.3% of control, n = 7, respectively). In contrast, isoflurane (1.4 vol% approximately 0.50 mM) preferentially depressed fEPSP amplitudes mediated by NMDA receptors (44.0 +/- 7.4% of control, n = 6, P < 0.001) compared with those for non-NMDA receptors (68.7 +/- 5.4% of control n = 6), indicating a selective, additional postsynaptic effect. Paired pulse facilitation of fEPSPs was increased significantly by both anesthetic agents from 1.37 +/- 0.13 to 1.91 +/- 0.25 (n = 5, P < 0.05 for halothane) and from 1.44 +/- 0.04 to 1.64 +/- 0.08 (n = 5, P < 0.01 for isoflurane), suggesting that presynaptic mechanisms are also involved in fEPSP depression produced by the anesthetic agents. Neither rise times nor decay times of fEPSPs were changed in the presence of the anesthetic agents. CONCLUSIONS: These results indicate that fEPSPs mediated by postsynaptic NMDA receptors are more sensitive to clinically relevant concentrations of isoflurane than are non-NMDA receptor-mediated responses, but this selective effect was not observed for halothane. Both agents also appeared to depress release of glutamate from nerve terminals via presynaptic actions.     

Monosynaptic connections between pairs of L5A pyramidal neurons in columns of juvenile rat somatosensory cortex

Layer 5 (L5) of somatosensory cortex is a major gateway for projections to intra- and subcortical brain regions. This layer is further divided into 5A and 5B characterized by relatively separate afferent and efferent connections. Little is known about the organization of connections within L5A of neocortical columns. We therefore used paired recordings to probe the anatomy and physiology of monosynaptic connections between L5A pyramidal neurons within the barrel columns of somatosensory cortex in acute slices of approximately 3-week-old rats. Post hoc reconstruction and calculation of the axodendritic overlap of pre- and postsynaptic neurons, together with identification of putative synaptic contacts (3.5 per connection), indicated a preferred innervation domain in the proximal dendritic region. Synaptic transmission was reliable (failure rate <2%) and had a low variability (coefficient of variation of 0.3). Unitary excitatory postsynaptic potential (EPSP) amplitudes varied 30-fold with a mean of 1.2 mV and displayed depression over a wide range of frequencies (2-100 Hz) during bursts of presynaptic firing. A single L5A pyramidal neuron was estimated to target approximately 270 other pyramidal neurons within the same layer of its home barrel column, suggesting a mechanism of feed-forward excitation by which synchronized single action potentials are efficiently transmitted within L5A of juvenile cortex.     

雌二醇诱导的慢性非细菌性前列腺炎大鼠前列腺平滑肌细胞电压依赖钙离子通道的定量表达

目的:建立慢性非细菌性前列腺炎大鼠模型,探讨炎症组与正常对照组大鼠前列腺平滑肌细胞电压依赖钙离子通道定量表达的差异性。方法:采用雌二醇诱导大鼠前列腺炎,体外培养纯化前列腺平滑肌细胞(PSMCs),用Trizol提取总RNA,经逆转录,SYBR Green I实时定量PCR测定各亚型钙离子通道α1亚基的mRNA相对表达水平,比较两组间的差异。结果:炎症组和对照组均见L、T、P/Q型钙通道表达,炎症组L型钙通道CaV1.2相对对照组表达量增多,分别为0.048±0.024和0.031±0.015,差异具有显著性(t=2.846,P=0.007),T、P/Q型两组间无统计学差异。结论:慢性前列腺炎PSMCs细胞膜L型钙通道CaV1.2数目上调,钙内流增多,在发病机制中可能具有一定作用。     

Immunephenotype of Glomerular and Interstitial Infiltrating Cells in Protocol Renal Allograft Biopsies and Histological Diagnosis

Patients with a protocol renal allograft biopsy simultaneously displaying interstitial fibrosis/tubular atrophy (IF/TA) and subclinical rejection (SCR) have a shortened graft survival than patients with a normal biopsy, or with a biopsy only displaying IF/TA or SCR. The poor outcome of these patients could be related with a more severe inflammation. We evaluate the immunophenotype of infiltrating cells in these diagnostic categories. Nonexhausted paraffin blocks from protocol biopsies done during the first year were stained with anti-CD45, CD3, CD20, CD68 and CD15 monoclonal antibodies. Glomerular and interstitial positive cells were counted. C4d deposition in peritubular capillaries was evaluated. Histological diagnoses were: normal (n = 80), SCR (n = 17), IF/TA (n = 42) and IF/TA + SCR (n = 17). Only interstitial CD20 positive cells were significantly increased in patients displaying IF/TA + SCR; normal (137 +/- 117), SCR (202 +/- 145), IF/TA (208 +/- 151) and IF/TA + SCR (307 +/- 180 cells/mm(2)), p < 0.01. The proportion of biopsies displaying C4d deposition was not different among groups. The upper tertile of CD20 positive interstitial cells was associated with a decreased death-censored graft survival (relative risk: 3.01, 95% confidence interval: 1.23-7.35; p = 0.015). These data suggest that B-cell interstitial infiltrates are associated with histological damage and outcome, but do not distinguish whether these infiltrates were the cause or the consequence of chronic tubulo-interstitial damage.     

Agent-selective Effects of Volatile Anesthetics on GABAA Receptor-mediated Synaptic Inhibition in Hippocampal Interneurons

Background: A relatively small number of inhibitory interneurons can control the excitability and synchronization of large numbers of pyramidal cells in hippocampus and other cortical regions. Thus, anesthetic modulation of interneurons could play an important role for the maintenance of anesthesia. The aim of this study was to compare effects produced by volatile anesthetics on inhibitory postsynaptic currents (IPSCs) of rat hippocampal interneurons.

Methods: Pharmacologically isolated [gamma]-aminobutyric acid type A (GABAA) receptor-mediated IPSCs were recorded with whole cell patch-clamp techniques in visually identified interneurons of rat hippocampal slices. Neurons located in the stratum radiatum-lacunosum moleculare of the CA1 region were studied. The effects of clinically relevant concentrations (1.0 rat minimum alveolar concentration) of halothane, enflurane, isoflurane, and sevoflurane were compared on kinetics of both stimulus-evoked and spontaneous GABAA receptor-mediated IPSCs in interneurons.

Results: Halothane (1.2 vol% [almost equal to] 0.35 mm), enflurane (2.2 vol% [almost equal to] 0.60 mm), isoflurane (1.4 vol% [almost equal to] 0.50 mm), and sevoflurane (2.7 vol% [almost equal to] 0.40 mm) preferentially depressed evoked IPSC amplitudes to 79.8 +/- 9.3% of control (n = 5), 38.2 +/- 8.6% (n = 6), 52.4 +/- 8.4% (n = 5), and 46.1 +/- 16.0% (n = 8), respectively. In addition, all anesthetics differentially prolonged the decay time constant of evoked IPSCs to 290.1 +/- 33.2% of control, 423.6 +/- 47.1, 277.0 +/- 32.2, and 529 +/- 48.5%, respectively. The frequencies of spontaneous IPSCs were increased by all anesthetics (twofold to threefold). Thus, the total negative charge transfer mediated by GABAA receptors between synaptically connected interneurons was enhanced by all anesthetics.     

Comparative study of the efficacy of the common topical hemostatic agents with fibrin sealant in a rabbit aortic anastomosis model

OBJECTIVE: The purpose of this study was to compare the hemostatic efficacy of the common surgical hemostatic agents with fibrin sealant (FS) and to assess their functional strength to secure hemostasis in lieu of placing additional sutures. METHODS: End-to-end anastomosis of transected abdominal aorta was performed in moderately anticoagulated rabbits using 4 or 6 interrupted sutures. The suture line was covered either with gauze alone ("untreated") or with gauze plus Gelfoam, Avitene, Surgicel, FloSeal, or FS, following which blood flow was restored. Blood loss was absorbed by gauze and measured. The surviving rabbits were recovered and the repaired vessel was examined histologically 4 weeks after operation. The investigators were blinded to the treatment groups. Aortic anastomoses using 8 or 12 sutures (untreated) were also performed. RESULTS: Untreated 4-suture anastomosis of aorta resulted in a profuse hemorrhage with an average 108.0 +/- 19.2 (mean +/- SD) ml blood loss and 100% mortality (n = 4). FS application sealed the anastomoses, prevented blood loss (P < 0.01 vs untreated) and exsanguination of the rabbits (n = 4). Other hemostatic agents reduced the bleeding to varying degrees compared to the untreated animals (Gelfoam 66.4 +/- 17.6, Avitene 80.6 +/- 34, Surgicel 66.7 +/- 16.7, FloSeal 44.2 +/- 8.5 ml blood loss, n = 4/group), but the changes were not statistically significant. One to three rabbits in each group survived the operation. Six-suture aortic anastomoses, untreated, resulted in 67.7 +/- 21.8 ml blood loss and 100% survival (n = 6). Application of FS produced immediate and sustained hemostasis in all the animals (P < 0.01 vs untreated). Other hemostatic agents also reduced the bleeding (Gelfoam 42.5 +/- 10, Avitene 50.9 +/- 12.4, Surgicel 32.1 +/- 14, FloSeal 33.9 +/- 5.4 ml blood loss, n = 6/group), but the changes were not statistically significant. The 8- and 12-suture aorta repairs resulted in a moderate blood loss (43.9 +/- 19 and 21.3 +/- 14.9 ml, respectively), followed by a stable hemostasis that precluded the need to use any hemostatic agent. The aortic cross-clamping time of the 12-suture and time to hemostasis for both the 8- and the 12-suture techniques were significantly longer than those of the 4-suture plus FS application (P < 0.01, P < 0.01 and P < 0.05, respectively). CONCLUSION: In a moderate coagulopathy, FS was proven to be the most efficacious hemostatic agent, producing immediate and sustained hemostasis at the arterial anastomotic site. This high efficacy is in part attributed to the strong tissue adhesive property of this agent. FS application may potentially ease the anastomosis and shorten the duration of timely critical vascular procedures.