Tail Nerve Electrical Stimulation and Electro-Acupuncture Can Protect Spinal Motor Neurons and Alleviate Muscle Atrophy after Spinal Cord Transection in Rats.

Spinal cord injury (SCI) often results in death of spinal neurons and atrophy of muscles which they govern. Thus, following SCI, reorganizing the lumbar spinal sensorimotor pathways is crucial to alleviate muscle atrophy. Tail nerve electrical stimulation (TANES) has been shown to activate the central pattern generator (CPG) and improve the locomotion recovery of spinal contused rats. Electroacupuncture (EA) is a traditional Chinese medical practice which has been proven to have a neural protective effect. Here, we examined the effects of TANES and EA on lumbar motor neurons and hindlimb muscle in spinal transected rats, respectively. From the third day postsurgery, rats in the TANES group were treated 5 times a week and those in the EA group were treated once every other day. Four weeks later, both TANES and EA showed a significant impact in promoting survival of lumbar motor neurons and expression of choline acetyltransferase (ChAT) and ameliorating atrophy of hindlimb muscle after SCI. Meanwhile, the expression of neurotrophin-3 (NT-3) in the same spinal cord segment was significantly increased. These findings suggest that TANES and EA can augment the expression of NT-3 in the lumbar spinal cord that appears to protect the motor neurons as well as alleviate muscle atrophy.

Emulsified isoflurane enhances thermal transient receptor potential vanilloid-1 channel activation-mediated sensory/nociceptive blockade by QX-314.

BACKGROUND: QX-314 produces nociceptive blockade, facilitated by permeation through transient receptor potential vanilloid-1 (TRPV1) channels. TRPV1 channel can be activated by noxious heat and sensitized by volatile anesthetics. The authors hypothesized that emulsified isoflurane (EI) could enhance thermal TRPV1 channel activation-mediated sensory/nociceptive blockade by QX-314. METHODS: Rats were perineurally injected with QX-314 (Sigma-Aldrich Co. Ltd. Shanghai, China) alone or QX-314 combined with EI, followed by heat exposure on the injection site. The tail-flick and tail-clamping tests were used to assess sensory and nociceptive blockade, respectively; a sciatic nerve block model was used to assess motor and sensory blockade. Effects of EI on thermal activation of TRPV1 channels were evaluated on rat dorsal root ganglia neurons by whole-cell patch-clamp recordings. RESULTS: Heat exposure enhanced sensory/nociceptive blockade by QX-314 in rat tails, but not motor blockade in sciatic nerve block model. QX-314 alone or QX-314 + 42°C produced no nociceptive blockade. QX-314 + 48°C produced 100% nociceptive blockade with duration of 12.5 ± 2.0 h (mean ± SEM). By adding 2% EI, QX-314 + 42°C produced 80% nociceptive blockade with duration of 8.1 ± 1.9 h, which was similar to the effect of QX-314 + 46°C (7.7 ± 1.1 h; P = 0.781). The enhancement of heat on sensory/nociceptive blockade of QX-314 was prevented by TRPV1 channel antagonist. The temperature thresholds of TRPV1 channel activation on dorsal root ganglia neurons were significantly reduced by EI. CONCLUSIONS: Thermal activation of TRPV1 channels enhanced long-lasting sensory/nociceptive blockade by QX-314 without affecting motor blockade. The addition of EI reduced temperature thresholds for inducing long-lasting sensory/nociceptive blockade due to QX-314.

High-fidelity evoked potential for mapping the rat tail in thalamus.

The technique of field potentials (FPs) provides a macroscopic view for exploring brain function, and is supplementary to single-unit recording, a microscopic view that investigates each neuron in great detail. Mapping the rat tail in the ventroposterolateral (VPL) nucleus of the thalamus was carried out by analyzing the current source density (CSD) of the evoked FPs. The results showed a clear somatotopic organization of the tail in the VPL nucleus. Also, to obtain high-fidelity FPs, two recording parameters were determined. Based upon cross-correlation coefficient (rho), the cycles of FPs needed to be averaged should not be less than 50 and the distance between the two recording sites should be no longer than 50 mum in each direction (mediolateral, anteroposterior and ventrodorsal). Under these conditions, the representation (or reproducibility) of an FP can be >95%. The procedures used to determine these parameters can serve as a guide to obtain FPs with high signal-to-noise ratio and without spatial aliasing error.

Thermal effect of sonophoresis for accelerating the analgesic effect of local anesthetics on rat tail nerve.

Sonophoresis is an ultrasound transdermal drugs delivery system. The eutectic mixture of local anesthetics (EMLA) has been used clinically for anesthesia but requires at least one hour to take effect and lacks of analgesia's objective assessment. We proposed that sonophoresis could reduce the duration of EMLA analgesia effect onset and be assessed by sensory conduction studies. Thirty Wistar adult rats were randomized into normal, control, ultrasound-, and heat-treatment groups. Normal group was received no EMLA cream or ultrasound and heat treatment. The control group received the EMLA cream on the rat tail at 3.5 cm distal to the rat tail base for local anesthesia of tail nerve. Ultrasound- and heat-treatment groups were received ultrasound with different parameters and heat treatment, respectively, before EMLA cream applied. Sensory conduction studies of tail nerve were made before and after treatment every 5 min at least for 60 min in all rats. There was no significant difference between the EMLA control group and heat treatment group. All rats in ultrasound-treatment group exhibit significant difference with EMLA control group and heat-treatment group in time for decreased 20% SNCV except for the 2 W/cm(2), 25 min, 20% in ultrasound-treatment group having no significant difference with heat-treatment group. There was no significant difference between ultrasound-treatment subgroups. In the decrease of amplitude, only the 2 W/cm(2), 5 min, 100% and the 2 W/cm(2), 10 min, 50% in ultrasound-treatment group had significant difference between EMLA control and heat-treatment groups. We have objectively examined the sonophoresis effect of ultrasound by investigating the effects of EMLA. Applying ultrasound for 5 min reduces the onset time of EMLA analgesia from 60 min to less than 20 min. Ultrasound sonophoresis of analgesic drugs is potentially useful in the treatment of carpal tunnel syndrome, tooth extraction, and other applications of analgesia.

Relieving effects of electroacupuncture on mechanical allodynia in neuropathic pain model of inferior caudal trunk injury in rat: mediation by spinal opioid receptors.

The relieving effects of electroacupuncture (EA) on mechanical allodynia and its mechanism related to the spinal opioid system were investigated in a rat model of neuropathic pain. To produce neuropathic pain in the tail, the right superior caudal trunk was resected between the S1 and S2 spinal nerves. Two weeks after the surgery, EA stimulation (2 or 100 Hz, 0.3 ms, 0.2-0.3 mA) was delivered to Zusanli (ST36) for 30 min. The degree of mechanical allodynia was evaluated quantitatively by touching the tail with von Frey hair (2.0 g) at 10 min intervals. These rats were then subjected to an i.t. injection with one of the three specific opioid agonists in successive ways: the mu agonist (DAMGO 25, 50 and 100 pmol), the delta agonist (DADELT II 0.5, 1 and 2 nmol), and the kappa agonist (U50488H 5, 10 and 20 nmol) separated by 10 min in cumulative doses. During 30 min of EA stimulation, specific opioid antagonists were subjected to i.t. injection: the mu antagonist (beta-FNA 5, 10 and 20 nmol), the delta antagonist (naltrindole 5, 10 and 20 nmol), and the kappa antagonist (nor-BNI 3, 6 and 12 nmol) separated by 10 min in cumulative doses. As a result, EA reduced the behavioral signs of mechanical allodynia. Two Hz EA induced a robust and longer lasting effect than 100 Hz. All three opioid agonists also showed relieving effects on mechanical allodynia. However, nor-BNI could not block the EA effects on mechanical allodynia, whereas beta-FNA or naltrindole significantly blocked EA effects. These results suggest that the mu and delta, but not kappa, opioid receptors in the spinal cord of the rat, play important roles in mediating relieving effects on mechanical allodynia induced by 2 Hz EA.

Cutaneous sympathetic motor rhythms during a fever-like response induced by prostaglandin E(1).

Neuronal population discharges within the CNS and in somatic and sympathetic motor nerves often display oscillations. Peripheral oscillations may provide a window into central mechanisms, as they often show coherence with population activity of subsets of central neurones. The reduction in heat loss through the cutaneous circulation during fever may be mediated via sympathetic premotor neurones not utilised during normal temperature regulation. Consequently, here we assessed, in anaesthetised rats, whether the frequency signature of population sympathetic discharge observed in neurones innervating the tail (thermoregulatory) circulation changed during a fever-like response induced by intracerebroventricular injection of prostaglandin E(1). We found that when core temperature was raised to 38.8-40.5 degrees C sympathetic activity was abolished. Following administration of prostaglandin (400 ng or 1 microg per rat), activity was restored to levels seen prior to heating (154+/-53.5%; n=10). Injection of vehicle had no effect (n=7). Prior to heating when most animals were in central apnoea (14/18) two peaks were observed in autospectra of sympathetic activity: one at 0.68-0.93 Hz (T-peak) and another at the frequency of ventilation (2 Hz). Central respiratory drive was recruited during hyperthermia where it was 1:2 locked to the frequency of ventilation and following prostaglandin administration, an additional peak in sympathetic autospectra was seen at this frequency. Time-evolving spectra indicated that this peak resulted from the dynamic locking of the 'T-peak' to central respiratory drive. Our data show that during a fever-like response the dominant oscillations in sympathetic activity controlling a thermoregulatory circulation and their dynamic coupling to respiratory-related inputs are similar to those seen under normal conditions. Therefore, during this fever-like response, the neural substrate(s) underlying the oscillations is not reconfigured and remains capable of sculpturing the pattern of sympathetic neuronal discharge that may be regulated by several descending pathways.

Respiratory rhythmicity in the activity of postganglionic neurones supplying the rat tail during hyperthermia.

It has been suggested that thermoregulatory stimulation changes respiration-related rhythmicity in the activity of postganglionic sympathetic neurones supplying the rat tail to a distinct modulation independent of respiration. To study this possibility, single and few fibre recordings were made from ten filaments split from the ventral collector nerves of the rat during whole body warming. Sympathetic activity was analysed by autocorrelation and phrenic-triggered summation. All neurones except one were gradually inhibited and lost their on-going activity above a core temperature of 39-39.5 degrees C while the frequency of the phrenic bursts increased significantly. During hyperthermia, all neurones tested exhibited a prominent respiratory modulation in their activity which, compared to normothermia, was significantly increased in strength, or even newly acquired. No other rhythm emerged. These results speak against the hypothesis that in the rat sympathetic pathways controlling the tail vasculature and thus involved in thermoregulation, during hyperthermia become controlled by central oscillators distinct from the respiratory rhythm generator. Rather, respiratory modulation appears to remain the dominant rhythm as is common for sympathetic neurones supplying other cardiovascular targets.

Tail-flick test: II. The role of supraspinal systems and avoidance learning.

It is held that the tail-flick test of pain depends on a spinal reflex because a similar response is observed in spinally transected rats. But when subjects were manually held and a cool heat setting was used, supraspinal systems facilitated the response (Experiment 1). This effect did not depend on the rate at which the tail was heated (Experiment 2) but rather on the co-occurrence of visual, auditory, and tactile cues that predict impending pain (Experiments 3 and 4). Subjects rapidly learned to exhibit a tail movement during these co-occurring cues, and this avoidance response was instrumental in nature (Experiment 5). Optimal learning was observed when the visual signal was presented 8-12 s before a heat-elicited response is normally observed (Experiment 6), and a low dose of morphine inhibited the performance of the instrumental response (Experiment 7).

Interneurons presynaptic to rat tail-flick motoneurons as mapped by transneuronal transport of pseudorabies virus: few have long ascending collaterals.

The method of transneuronal retrograde transport of the Bartha strain of the swine alpha-herpes virus, pseudorabies virus, was used to identify putative interneurons presynaptic to motoneurons that supply a tail-flick muscle in the rat. We also investigated whether these interneurons also contribute to ascending somatosensory pathways. Two to five days after injection of pseudorabies virus into the left abductor caudae dorsalis muscle, and cholera toxin B into the right somatosensory thalamus and midbrain, rats were perfused and spinal cord sections processed immunohistochemically in a two-step procedure to stain cholera toxin B-immunoreactive cells black and pseudorabies virus-immunoreactive cells brown. At short (two-day) survivals, the first spinal neurons to be pseudorabies virus-immunoreactive were in the ipsilateral abductor caudae dorsalis motoneuron pool (S3-S4) and intermediolateral cell column (T12-L2), with a few (0 to five/section) bilaterally in the intermediate zone and around the central canal (all lumbosacral levels). With longer (three- to four-day) survival, more cells were noted (20-50/section) bilaterally (ipsilateral preponderance) in the dorsal and ventral horns of the lumbosacral cord. Many were in lamina I (marginal layer), while few were in lamina II (substantia gelatinosa). At four- and five-day survivals, the numbers of cells increased (20 to 100/section) bilaterally and now included lamina II. The fact that unilateral rhizotomy at L4-Co1 failed to change the distribution of spinal pseudorabies virus labeling suggests that the labeling was due to retrograde transport via the ventral root. In support, bilateral removal of the lumbar sympathetic ganglia, which receive their preganglionic innervation through the ventral root, reduced pseudorabies virus immunoreactivity throughout the thoracic and rostral lumbar spinal cord. These data indicate that there are (i) direct projections from intermediate and dorsal horn cells to abductor caudae dorsalis motoneurons, and (ii) disynaptic connections from dorsal horn (possibly including lamina II) cells to more ventral last-order interneurons. We also suggest that some lamina II cells are presynaptic to lamina I cells that project directly to abductor caudae dorsalis motoneurons. We observed cholera toxin B-immunoreactive cells (five to 20/section) in the expected locations (contralateral lamina I, deep dorsal horn and intermediate zone; lateral spinal nucleus bilaterally). Double-labeled (i.e. pseudorabies virus- and cholera toxin B-immunoreactive) neurons were only occasionally seen in the lateral spinal nucleus and were absent in the spinal gray matter, indicating that segmental interneurons do not collateralize in long ascending sensory pathways to the midbrain and somatosensory thalamus.

Diencephalon as a cardinal neural structure for mediating 2 Hz- but not 100 Hz-electroacupuncture-induced tail flick reflex suppression.

Transections at two levels of the forebrain were undertaken in the rat to investigate the neural structures indispensable for the mediation of tail flick reflex suppression (TFRS) induced by low (2 Hz) and high (100 Hz) frequency electroacupuncture (EA) stimulation. Removal of the telencephalic structures did not affect TFRS produced by high frequency EA, although a mild and temporal attenuation was observed for low frequency EA-induced TFRS. Ablation of the whole forebrain (the telencephalon and diencephalon) resulted in a total abolishment of 2-Hz EA effect as measured 5, 24 and 72 h after surgery. In the meantime there was a moderate attenuation (-32.8%, P less than 0.05) of 100-Hz EA effect, which appeared 5 h after the operation and recovered after 24 h. The results indicate that (1) high frequency EA effect persisted in animals devoid of the whole forebrain structures; (2) an intact diencephalon is indispensable for the neural circuitry controlling low frequency EA-induced TFRS.

Behavioral and thalamic nociceptive responses in rats following noxious ischaemia of the tail.

In rats anaesthetized with urethane, we have investigated the response of neurones in the ventrobasal complex of the thalamus to noxious ischaemia of the tail, and to graded noxious thermal stimulation of the tail before and after ischaemia. In behavioural experiments conscious rats were exposed to the same experimental procedure. After ischaemia the threshold tail temperature required to elicit both a neuronal response and aversive behaviour in conscious rats, to thermal stimulation, was decreased significantly (P less than 0.01 paired t tests). Threshold temperatures for the neuronal response and the behavioural response were not significantly different, either before or after ischaemia. The time course of recovery to pre-ischaemic threshold temperatures was the same for both the behavioural and neuronal responses. Most thalamic neurones responding to noxious thermal stimulation of the tail also increased firing rate during ischaemia. The latency of response of the thalamic neurones to ischaemia was 12.1 +/- 1.8 min and the latency of the behavioural response to the same stimulus was 11.9 +/- 2.1 min. Ventrobasal thalamic neurones, therefore, which responded to noxious thermal stimulation of the tail also responded to noxious ischaemia, and exhibited a neuronal correlate of post-ischaemic hyperalgesia which paralleled behavioural responses closely.

Conduction block in a branching axon innervating two muscles under physiological conditions.

The escape reflex of the lobster consists of a series of tail flips resulting from alternating activity of the abdominal flexor and extensor muscles. Electromyographic (EMG) activity was recorded from the medial (DEAM) and the lateral (DEAL1) deep abdominal extensor muscles during free swimming. During the escape response, the muscles were active either synchronously or separately, at frequencies of 100-120 Hz. This activity pattern could be generated either by central programming, or by a peripheral mechanism such as frequency-dependent differential conduction block into one of the two branches of the common excitor axon (C.Ex) innervating these muscles. In order to explore the latter possibility in a living animal, we left the DEAM and DEAL1 muscles innervated only by the C.Ex from the tested segment. This was accomplished by manually cutting all other axons in the nerve under visual control. During escape responses in six successfully dissected animals, we found 27 sudden failures of the DEAM responses and only three in DEAL1. The failures were usually preceded by an increase in the delay of the response. These findings strongly suggest that conduction block occurs in the M branch innervating the DEAM under physiological conditions.

Alterations in the ipsi- and contralateral afferent inputs of dorsal horn cells produced by capsaicin treatment of one sciatic nerve in the rat.

Dorsal horn cells in the lumbar spinal cord of decerebrate spinal rats were examined 7-21 days following local application of capsaicin to the sciatic nerve. Such local capsaicin treatment is known not to influence the size of the incoming A and C fibre afferent volley. The receptive field properties and primary afferent input of cells on both sides of the cord, that is ipsi and contralateral to the treated nerve, were examined. On the treated side, the percentage of cells excited by C fibres from the capsaicin treated nerve was 30% of normal and the number of cells responding to noxious heating of the cutaneous receptive field was reduced by 50%. A fibre input and low and high threshold mechanical input were normal. The receptive field size was larger in many cells innervated by the treated nerve. On the side opposite to the treated nerve, responses to noxious and non-noxious stimulation of the untreated limb were unaffected as was the input from the untreated sciatic nerve. Receptive fields were somewhat larger than normal. Effects were also observed from contralateral stimuli. Cells on both sides of the cord were found with excitatory contralateral receptive fields and excitatory responses to trains of high intensity stimulation to the contralateral sciatic nerve. In untreated animals the effect of such contralateral stimulation is inhibitory. The results show that peripheral nerve capsaicin treatment causes long lasting reduction of the C fibre input to dorsal horn cells on the treated side. However, it also results in changes in the inhibitory and excitatory receptive fields of cells on both sides of the cord.