Isoflurane Suppresses the Excitability and Synaptic Transmission of Spinal Nociceptive Pathway in Rats

This is an open-access article, published by Evidence Based Communications (EBC). This work is licensed under the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium or format for any lawful purpose.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. From Department of Anesthesiology and Pain Medicine, Xijing Hospital, Fourth Military Medical University, Xi'an, China.

I soflurane (IFL) is one of the most widely used volatile anesthetics for its rapidly induction, circulatory stability, excellent muscle relax and less side effects (1,2). It was generally assumed that IFL induces anesthesia through potentiating the function of gamma-aminobutyric acid (GABA) A receptors (3), increasing the inhibitory neurotransmitter content such as GA-BA, and reducing the content of acetylcholine and other excitatory neurotransmitters (3)(4)(5). Animal studies demonstrated that IFL also acts on N-methyl-D -aspartic acid (NMDA) receptors (6)(7)(8), the two pore domain potassium background channels (9), voltage-gated calcium (10) and sodium channels (11).
The substantia gelatinosa (SG, lamina II) of the spinal superficial dorsal horn is consisted of several neuronal circuits involving different types of excitatory and inhibitory neurons that receive synaptic inputs from primary unmyelinated C and thinly myelinated A fibers. The spinal SG is particularly rich of molecules that act as synaptic mediators to influence neuronal excitability and synaptic transmission. Therefore, it is generally accepted that the spinal SG neu- Original Article rons play an important part in the transmission and modulation of nociceptive signals (22)(23)(24).
To further investigate the mechanisms underlying the analgesic action of IFL, the present study utilized patch-clamp technique to test the hypothesis that IFL can inhibit the monosynaptic events from primary C and Aδ fibers to SG neurons, and suppress the action potential discharge of SG neurons.

Ethics and Animals
Male Sprague Dawley (SD) rats (weighing 90 ± 10 g, 4-6 weeks postpartum) were provided by the Fourth Military Medical University Experimental Animal Center. The animal experiments were approved by the Institutional Animal Management Committee of Fourth Military Medical University. Animals were kept under controlled laboratory conditions: temperature 23-25℃ , relative humidity 58-62% , a light/dark cycle 12:12 hours and free access to food and water.

Drug Application
Concentration of IFL was given as volume percent (3% ) by passing the gas mixture (flow rate, 0.5 L/minute) into equilibrated ACSF through the respective vaporizers (Penlon Limietd, UK) for at least 15 minutes at room temperature (24 ± 1℃ ) (28). Solutions with IFL were contained in pyrex erlenmeyer flasks covered tightly with parafilm. The solubility of IFL at room temperature in ACSF is approximately two fold greater than at 37℃ . The concentration of IFL used in this study was equal approximately to twice the concentration of in vivo anesthesia (29,30).
Voltage-clamp whole-cell recordings were obtained from SG neurons by holding neurons at -70 mV to record excitatory postsynaptic currents (EPSCs) events. The firing pattern of neurons was tested in current clamp model with holding potential of -60 mV with 1 second long depolarizing pulses from the recording electrode. The attached dorsal root was stimulated by a suction electrode which connected with a stimulator (S88X Stimulator, Astro-Med, West Warwick, RI, USA). Graded 0.1-0.5 ms pulses stimulation of dorsal root was applied to evoke EPSCs (eEPSCs) in SG neurons. If dorsal root evoked response had a constant conduction velocities in several trials, repetitive stimulation (20 Hz for Aδ fiber response; 1 Hz for C fiber response) was used to further judge the monosynaptic or polysynaptic responses (22,23). Each eEPSC was recorded for 5 minutes, averaged from 15 eEPSCs sweeps. Action potentials evoked by depolarizing current injection were recorded under current clamp mode with clamping current 0 pA. Series resistances typically were less than 10 MΩ and were monitored throughout the recording period. Recording from a cell was discarded if the series resistance changed significantly (± 25% ) and the original value could not be recovered by manipulation of suction applied to the recording pipette. For reasons of recording stability, series resistance compensation was not used. Because series resistance was at least an order of magnitude less than the cell input resistances, voltage or current measurement errors attributable to series resistance were presumed to be less than 10% (22,23).
Axopatch 200B amplifier (Axon instruments, Union City, CA, USA), a Digidata 1440A (Axon instruments, Union City, CA, USA) and pClamp-fit10 software (Axon instruments, Union City, CA, USA) were used for data acquisition and analysis. The entire recording process was made at room temperature (24 ± 1℃ ). Chemical compounds used in this study were obtained from Sigma Company.

Characterization of Fiber Types
The types of primary fibers forming monosynaptic connection with SG neurons were categorized by the conduction velocity according to the latency and conduction distance. Details for the characterization were described in our previous publications (3,31,32). In this study, the Aδ and C fiber mediated eEPSCs were evoked by the intensity of stimulation at 0.98 ± 0.52 V and 5.13 ± 1.45 V, respectively. The conduction velocities of Aδ and C fibers were 1.37 ± 0.42 m/s and 0.28 ± 0.03 m/s, respectively, which were consistent with our previous studies (3,31,32).

Statistical Analysis
Prism 6.0 software was used to the statistical data analysis and plotting, including the test of normality of the distribution and homogeneity of variance. Data were presented as means ± standard deviation (SD). The difference of eEPSCs amplitude changes before and after dosing was analyzed by paired t-test. Difference was defined as significant at P<0.05.

IFL Significantly Inhibited the Peak Amplitude of Monosynaptic eEPSCs Mediated by Primary Aδ Fibers
Ten minutes after stable whole-cell recordings, Aδ fiber-mediated monosynaptic eEPSCs were recorded as control. After superfusion with IFL equilibrated ACSF for 1 minute, the amplitude of monosynaptic eEPSCs was significantly inhibited about 37.24 ± 14.31% , compared with controls (**P<0.01, IFL vs control; P>0.05, Wash vs control; N=8). This inhibitory effect could be washed out by continuous perfusion of normal ACSF for about 10 minutes (Figure 1) , suggesting that the suppressive effect of IFL on synaptic transmission from Aδ fibers to SG neurons is reversible in a short time schedule.

IFL Significantly Suppressed C Fiber-Mediated Monosynaptic eEPSCs
The whole cell recordings were performed as same as experiment one, the membrane potential was held at -70 mV. The segmental dorsal root was stimulated by a suction electrode. When the C fiber-mediated monosynaptic eEP-SCs were challenged by IFL, the peak amplitude of eEPSCs was suppressed by 28.67 ± 1.05% (***P<0.001, IFL vs control; P>0.05, Wash vs control; N=7). The suppressive effect of IFL on synaptic transmission from C fibers to SG neu- rons is also reversible (Figure 2).

IFL Suppressed the Action Potential Firing Frequency of SG Neurons
To further examine the effect of IFL on the excitability of SG neurons, the whole-cell recordings were switched to current clamp mode. Depolarizing current injection to recorded neurons was used to initiate action potentials. Tonic (Figure 3, upper panel) and phasic firing (Figure 3, lower panel) patterns were evidenced in most re-corded SG neurons, only a few neurons expressed delayed firing pattern (data not shown). Perfusion IFL equilibrated normal ACSF for 1 minute significantly decreased the firing frequency of SG neurons. The numbers of APs decreased from 7.28 ± 1.09 to 3.13 ± 1.33 in tonic firing neurons (***P<0.001, IFL vs control; P> 0.05, Wash vs control; N=10, Figure 3, upper panel), from 8.09 ± 2.01 to 3.12 ± 2.03 in phasic firing neurons (***P<0.001, IFL vs control; P> 0.05, Wash vs control; N=9, Figure 3, lower panel) and from 4 to 2 in delayed firing neurons (**P<0.01, IFL vs control; P>0.05, Wash vs control; N=2; data not shown). These effects were reversible in all cases (Figure 3).

DISCUSSION
Superficial dorsal horn is the spinal center for pain transmission and modulation, which receives the peripheral nociceptive information mainly through primary C and Aδ fibers (33). Electrical stimulation to primary nociceptive afferents can imitate different intensity of pain signals. The present electrophysiological study demonstrated that IFL can reduce the excitability of SG neurons and suppress the synaptic transmission from primary nociceptive fibers to SG neurons in the spinal dorsal horn, suggesting that IFL plays analgesic role through inhibiting or weakening the excitability and synaptic transmission of spinal nociceptive pathway. An earlier study has used the similar recording techniques and examined the effect of IFL on the spinal excitatory synaptic transmission at 37℃ (34). They did find the suppressive action of IFL on the excitatory polysynaptic responses in SG neurons possibly through the potentiation of the inhibitory synaptic transmission, but failed to detect the direct effects on excitatory monosynaptic responses. Because the solubility of IFL significantly depends on the environmental temperature, the solubility of IFL at room temperature (24℃ ) is approximately two fold greater than at 37℃ . The concentration of IFL used in the present study is about 3% , two fold greater than the previous study (1.5% ) (29,30,34). Therefore, the different concentration of IFL may be responsible for the discrepancies between our study and the earlier study.
Original Article   Most studies concerning the effect of IFL on neuronal excitability and synaptic transmission were conducted in the brain neurons. Antkowiak and Helfrich-Forster (15) have reported that IFL can enhance the central inhibitory synaptic response which was mediated by GABA receptors. Jones and Harrison found that IFL can extend GABA receptor-mediated inhibitory postsynaptic currents in cultured rat hippocampal neurons (16). Extracellular recording of hippocampal CA1 neurons showed that 2.0% IFL can reduce NMDA receptor-mediated current (17).
Several studies revealed that IFL can reduce the excitability of central neurons. IFL can increase the chloride ion flow (13,22),decrease membrane resistance via activation of the two pore leak potassium channels (35,36), and finally leads to the hyperpolarization of central neuronal membrane and decrease of action potential discharges. Our data suggested that IFL can significantly reduce the action potential firing frequency of SG neurons, which was consistent with the findings obtained from central neurons.
Recently, the spinal mechanisms of IFL in-duced analgesia have become a new research focus. Zhou et al. (18) reported that emulsified IFL can produce regional anesthesia by inhibiting of voltage-gated Na + channels, and enhance nociceptive blockade of QX-314 by thermal transient receptor potential vanilloid 1 (TRPV1) channel activation in the spinal level (19,37). The limitation of present study would be the nature of in vitro recordings. The isolated spinal cord slices were maintained in a somewhat artificial environment. For instance, the room temperature is much lower than normal physical condition. Therefore, the effects of IFL revealed in this study may not be a mimesis of in vivo experiments. Further in vivo study is needed.
In conclusion, the present study illuminated that IFL may directly inhibit the excitability and synaptic transmission of spinal nociceptive pathway, and may contribute to the analgesia action.