Rat Dorsal Horn Neurons Research Articles

Spinophilin modulates pain through suppressing dendritic spine morphogenesis via negative control of Rac1-ERK signaling in rat spinal dorsal horn

Both spinophilin (SPN, also known as neurabin 2) and Rac1 (a member of Rho GTPase family) are believed to play key roles in dendritic spine (DS) remodeling and spinal nociception. However, how SPN interacts with Rac1 in the above process is unknown. Here, we first demonstrated natural existence of SPN-protein phosphatase 1-Rac1 complex in the spinal dorsal horn (DH) neurons by both double immunofluorescent labeling and co-immunoprecipitation, then the effects of SPN over-expression and down-regulation on mechanical and thermal pain sensitivity, GTP-bound Rac1-ERK signaling activity, and spinal DS density were studied. Over-expression of SPN in spinal neurons by intra-DH pAAV-CMV-SPN-3FLAG could block both mechanical and thermal pain hypersensitivity induced by intraplantar bee venom injection, however it had no effect on the basal pain sensitivity. Over-expression of SPN also resulted in a significant decrease in GTP-Rac1-ERK activities, relative to naive and irrelevant control (pAAV-MCS). In sharp contrast, knockdown of SPN in spinal neurons by intra-DH pAAV-CAG-eGFP-U6-shRNA[SPN] produced both pain hypersensitivity and dramatic elevation of GTP-Rac1-ERK activities, relative to naive and irrelevant control (pAAV-shRNA [NC]). Moreover, knockdown of SPN resulted in increase in DS density while over-expression of it had no such effect. Collectively, SPN is likely to serve as a regulator of Rac1 signaling to suppress DS morphogenesis via negative control of GTP-bound Rac1-ERK activities at postsynaptic component in rat DH neurons wherein both mechanical and thermal pain sensitivity are controlled.

Rat dorsal horn neurons primed by stress develop a long-lasting manifest sensitization after a short-lasting nociceptive low back input

A single injection of nerve growth factor (NGF) into a low back muscle induces a latent sensitization of rat dorsal horn neurons (DHNs) that primes for a manifest sensitization by a subsequent second NGF injection. Repeated restraint stress also causes a latent DHN sensitization. In this study, we investigated whether repeated restraint stress followed by a single NGF injection causes a manifest sensitization of DHNs. Rats were stressed repeatedly in a narrow plastic restrainer (1 hour on 12 consecutive days). Control animals were handled but not restrained. Two days after stress paradigm, behavioral tests and electrophysiological in vivo recordings from single DHNs were performed. Mild nociceptive low back input was induced by a single NGF injection into the lumbar multifidus muscle just before the recording started. Restraint stress slightly lowered the low back pressure pain threshold (Cohen d = 0.83). Subsequent NGF injection increased the proportion of neurons responsive to deep low back input (control + NGF: 14%, stress + NGF: 39%; P = 0.041), mostly for neurons with input from outside the low back (7% vs 26%; P = 0.081). There was an increased proportion of neurons with resting activity (28% vs 55%; P = 0.039), especially in neurons having deep input (0% vs 26%; P = 0.004). The results indicate that stress followed by a short-lasting nociceptive input causes manifest sensitization of DHNs to deep input, mainly from tissue outside the low back associated with an increased resting activity. These findings on neuronal mechanisms in our rodent model suggest how stress might predispose to radiating pain in patients.

L-bupivacaine Inhibition of Nociceptive Transmission in Rat Peripheral and Dorsal Horn Neurons.

Although the widely used single L-enantiomers of local anesthetics have less toxic effects on the cardiovascular and central nervous systems, the mechanisms mediating their antinociceptive actions are not well understood. The authors hypothesized that significant differences in the ion channel blocking abilities of the enantiomers of bupivacaine would be identified. The authors performed electrophysiologic analysis on rat dorsal root ganglion neurons in vitro and on spinal transmissions in vivo. In the dorsal root ganglion, these anesthetics decreased the amplitudes of action potentials. The half-maximum inhibitory concentrations of D-enantiomer D-bupivacaine were almost equal for Aβ (29.5 μM), Aδ (29.7μM), and C (29.8 μM) neurons. However, the half-maximum inhibitory concentrations of L-bupivacaine was lower for Aδ (19.35 μM) and C (19.5 μM) neurons than for A β (79.4 μM) neurons. Moreover, D-bupivacaine almost equally inhibited tetrodotoxin-resistant (mean ± SD: 15.8 ± 10.9% of the control, n = 14, P < 0.001) and tetrodotoxin-sensitive (15.4 ± 15.6% of the control, n = 11, P = 0.004) sodium currents. In contrast, L-bupivacaine suppressed tetrodotoxin-resistant sodium currents (26.1 ± 19.5% of the control, n = 18, P < 0.001) but not tetrodotoxin-sensitive sodium currents (74.5 ± 18.2% of the control, n = 11, P = 0.477). In the spinal dorsal horn, L-bupivacaine decreased the area of pinch-evoked excitatory postsynaptic currents (39.4 ± 11.3% of the control, n = 7, P < 0.001) but not touch-evoked responses (84.2 ± 14.5% of the control, n = 6, P = 0.826). In contrast, D-bupivacaine equally decreased pinch- and touch-evoked responses (38.8 ± 9.5% of the control, n = 6, P = 0.001, 42.9 ± 11.8% of the control, n = 6, P = 0.013, respectively). These results suggest that the L-enantiomer of bupivacaine (L-bupivacaine) effectively inhibits noxious transmission to the spinal dorsal horn by blocking action potential conduction through C and Aδ afferent fibers.

Action potentials and subthreshold potentials of dorsal horn neurons in a rat model of myositis: a study employing intracellular recordings in vivo.

Intracellular in vivo recordings from rat dorsal horn neurons were made to study the contribution of microglia to the central sensitization of spinal synapses induced by a chronic muscle inflammation. To block microglia activation, minocycline was continuously administered intrathecally during development of the inflammation. The aim was to test whether an inflammation-induced sensitization of dorsal horn neurons is mediated by changes in synaptic strength or other synaptic changes and how activated microglia influence these processes. Intracellular recordings were used to measure subthreshold excitatory postsynaptic potentials (EPSPs) and suprathreshold action potentials (APs). The muscle inflammation significantly increased the proportion of dorsal horn neurons responding with APs or EPSPs to electrical stimulation of the muscle nerve from 27 to 56% (P < 0.01) and to noxious muscle stimulation (3 vs. 44%, P < 0.01). Neurons showing spontaneous ongoing AP or EPSP activity increased from 28 to 74% (P < 0.01). Generally, the increases in suprathreshold AP responses did not occur at the expense of subthreshold EPSPs, because EPSP-only responses also increased. Intrathecal minocycline prevented the inflammation-induced increase in responsiveness to electrical (24%, P < 0.02) and mechanical stimulation (14%, P < 0.02); the effect was stronger on suprathreshold APs than on subthreshold EPSPs. The increase in ongoing activity was only partly suppressed. These data suggest that the myositis-induced hypersensitivity of the dorsal horn neurons to peripheral input and its prevention by intrathecal minocycline treatment were due to both an increase in the number of active synapses and an increased synaptic strength.NEW & NOTEWORTHY During a chronic muscle inflammation (myositis), activated microglia controls both the increase in the number of active synapses and the increase in synaptic strength.

Effect of remifentanil on iron metabolism in spinal dorsal horn neurons of rats

Objective To evaluate the effect of remifentanil on iron metabolism in spinal dorsal horn neurons of rats. Methods The primary spinal dorsal horn neurons of rats were seeded in the culture plate at a density of 2×105 cells/well and divided into 4 groups using a random number table method: control group (C group, n=40), remifentanil group (R group, n=40), divalent metal transporter 1 without iron-responsive element [DMT1(-)IRE] siRNA group (siRNA group, n=32) and DMT1(-)IRE siRNA plus remifentanil group (siRNA+ R group, n=32). siRNA and siRNA+ R groups were subjected to DMT1(-)IRE siRNA transfection on day 3 of culture.R and siRNA+ R groups were incubated for 60 min in the solution with remifentanil at a final concentration of 40 nmol/L.The contents of reactive oxygen species (ROS) and Fe2+ were determined by fluorescent probe method, the malondialdehyde(MDA)content was detected by TBA method, and the content of labile iron pool (LIP) was detected by calcein AM and iron chelator at the end of incubation with remifentanil in R and siRNA+ R groups and at the corresponding time points in the other groups.The expression of DMT1(-)IRE and DMT1(+ )IRE was determined by Western blot in C and R groups. Results Compared with C group, the expression of DMT1(-)IRE in the spinal dorsal horn neurons was significantly up-regulated, the contents of Fe2+, LIP, ROS and MDA were increased (P 0.05). Compared with R group, the contents of Fe2+, LIP, ROS and MDA in the spinal dorsal horn neurons were significantly decreased in siRNA+ R group (P<0.05). Conclusion Remifentanil increases the iron content of spinal dorsal horn neurons by activating DMT1(-)IRE, which may be associated with the mechanism of remifentanil-induced postoperative hyperalgesia in rats. Key words: Piperidines; Hyperalgesia; Iron; Spinal cord

Contributions of mTOR Activation-Mediated Upregulation of Synapsin II and Neurite Outgrowth to Hyperalgesia in STZ-Induced Diabetic Rats.

Painful diabetic neuropathy (PDN) is among the common complications in diabetes mellitus (DM), with its underlying mechanisms largely unknown. Synapsin II is primarily expressed in the spinal dorsal horn, and its upregulation mediates a superfluous release of glutamate and a deficiency of GABAergic interneuron synaptic transmission, which is directly implicated in the facilitation of pain signals in the hyperalgesic nociceptive response. Recently, synapsin II has been revealed to be associated with the modulation of neurite outgrowth, whereas the process of this neuronal structural neuroplasticity following neuronal hyperexcitability still remains unclear. In this study, we found that under conditions of elevated glucose, TNF-α induced the activation of mTOR, mediating the upregulation of synapsin II and neurite outgrowth in dorsal horn neurons. In vivo, we demonstrated that mTOR and synapsin II were upregulated and coexpressed in the spinal dorsal horn neurons in rats with streptozotocin (STZ)-induced diabetes. Furthermore, the intrathecal administration of the mTOR inhibitor rapamycin or synapsin II shRNA significantly diminished the expression of synapsin II, effectively mitigating hyperalgesia in PDN rats. We are the first to discover that in STZ-induced diabetic rats the activation of mTOR mediates the upregulation of synapsin II and neurite outgrowth, both contributing to hyperalgesia. These findings may benefit the clinical therapy of PDN by provision of a novel target.

The paracetamol metabolite N-acetylp-benzoquinone imine reduces excitability in first- and second-order neurons of the pain pathway through actions on KV7 channels.

Paracetamol (acetaminophen, APAP) is one of the most frequently used analgesic agents worldwide. It is generally preferred over nonsteroidal anti-inflammatory drugs because it does not cause typical adverse effects resulting from the inhibition of cyclooxygenases, such as gastric ulcers. Nevertheless, inhibitory impact on these enzymes is claimed to contribute to paracetamols mechanisms of action which, therefore, remained controversial. Recently, the APAP metabolites N-arachidonoylaminophenol (AM404) and N-acetyl-p-benzoquinone imine (NAPQI) have been detected in the central nervous system after systemic APAP administration and were reported to mediate paracetamol effects. In contrast to nonsteroidal anti-inflammatory drugs that rather support seizure activity, paracetamol provides anticonvulsant actions, and this dampening of neuronal activity may also form the basis for analgesic effects. Here, we reveal that the APAP metabolite NAPQI, but neither the parent compound nor the metabolite AM404, reduces membrane excitability in rat dorsal root ganglion (DRG) and spinal dorsal horn (SDH) neurons. The observed reduction of spike frequencies is accompanied by hyperpolarization in both sets of neurons. In parallel, NAPQI, but neither APAP nor AM404, increases currents through KV7 channels in DRG and SDH neurons, and the impact on neuronal excitability is absent if KV7 channels are blocked. Furthermore, NAPQI can revert the inhibitory action of the inflammatory mediator bradykinin on KV7 channels but does not affect synaptic transmission between DRG and SDH neurons. These results show that the paracetamol metabolite NAPQI dampens excitability of first- and second-order neurons of the pain pathway through an action on KV7 channels.

Spinal Cord Stimulation With “Conventional Clinical” and Higher Frequencies on Activity and Responses of Spinal Neurons to Noxious Stimuli: An Animal Study

Spinal cord stimulation (SCS) at both conventional and higher frequencies may effectively reduce pain, but optimal parameters need to be established. This study investigated how SCS at different frequencies and pulse widths acutely modulates nociceptive activity of wide dynamic range (WDR) and high threshold (HT) dorsal horn neurons in rats at a stimulus amplitude that influences both local circuits and dorsal column fibers. L2 -L3 and L6 -S2 spinal segments were exposed for SCS and spinal neuronal recordings, respectively. Responses to pinch of a hindpaw were recorded before and after SCS (40 or 200 µsec pulse width at 50, 500, 1 kHz and 10 kHz, amplitude: 90% of motor threshold) for 5 or 20 min. Pinch responses were tested within 30 s after SCS ceased (first pinch) and at ∼4 min intervals until response recovery. 1) SCS for 5 min suppressed averaged first pinch responses, except for 40 µsec/50 Hz. 2) Only SCS with 40 µs/1 kHz suppressed more spinal neurons than 200 µsec/50 Hz. 3) All SCS parameters at 5 min increased pinch responses for a small population of cells, with the incidence being greater for WDR than for HT neurons. 4) SCS at 1 kHz (40 or 200 µsec) for 20 min reduced the response to the second pinch as compared with baseline responses. In addition, no neurons exhibited increased pinch responses. Compared with a typical low frequency SCS (200 µs/50 Hz) or high-frequency SCS at 10 kHz, at an amplitude designed to influence both local spinal circuits and dorsal column fiber tracts, 1 kHz SCS suppressed nociceptive responses of more spinal neurons and/or demonstrated longer persisting suppressive effects. SCS at 1 kHz surpassed both low-frequency (50Hz) and high-frequency (10 kHz) SCS application in this normal animal model.

Short-term swimming exercise attenuates the sensitization of dorsal horn neurons in rats with NGF-induced low back pain.

Physical exercise has been shown to be an effective therapy for non-specific low back pain. The study investigated if swimming exercise is a means to reduce the spinal sensitization in an animal model of non-specific low back pain. In deeply anesthetized rats, dorsal horn neurons were recorded in spinal segment L2. To induce sensitization of dorsal horn neurons, two injections of nerve growth factor were made into the lumbar multifidus muscle at an interval of 5days. Swimming exercise for 30min was performed on the 5days between both NGF injections. A control group received the NGF injections without exercise treatment. Swimming exercise caused a significant decrease in the NGF-induced hyperexcitability of dorsal horn neurons. Compared to control, the proportion of neurons with input from deep somatic tissues and of convergent neurons with input from at least two types of different tissues decreased significantly (50% vs. 25% and 37% vs. 15%; both p<0.05). Swimming exercise also reduced the NGF-induced increase in neuronal resting activity. Both the proportion of active neurons and the mean discharge frequency of all neurons decreased significantly (60%, 76.3±23.1imp/min; vs. 25%, 51.7±35.1imp/min; both p<0.01). In our animal model of low back pain, short-term swimming exercise effectively reduced the latent sensitization of spinal dorsal horn neurons. Swimming exercise decreased the hyperexcitability of the neurons to low back input and lowered the resting activity of sensitized neurons. Physical exercise is a common treatment for low back pain. The possible mechanisms underlying the effects of exercise are probably multifold. This work shows that swimming exercise prevents sensitization of dorsal horn neurons, which may be one mechanism for the positive effects of exercise.

CB1 Receptors Mediated Inhibition of ATP-Induced [Ca2+]i Increase in Cultured Rat Spinal Dorsal Horn Neurons.

Spinal cannabinoid receptor 1 (CB1R) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CB1R and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB1 receptor activation modulates the function of P2X receptor channels within dorsal horn. For this reason, we observed the effect of CP55940 (cannabinoid receptor agonist) on ATP-induced Ca2+ mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca2+]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100μM ATP caused [Ca2+]i increase in cultured DH neurons. ATP-evoked [Ca2+]i increase in DH neurons was blocked by chelating extracellular Ca2+ and P2 purinoceptor antagonist PPADS. At the same time, ATP-γ-S (a non-hydrolyzable ATP analogue) mimicked the ATP action, while P2Y receptor agonist ADP failed to evoke [Ca2+]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca2+]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca2+ mobilization was inhibited after pretreated with CP55940 with a concentration-dependent manner, which implies that the opening of P2X receptor channels are down-regulated by activation of cannabinoid receptor. The inhibitory effect of CP55940 on ATP-induced Ca2+ response was mimicked by ACEA (CB1R agonist), but was not influenced by AM1241 (CB2R agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca2+ mobilization was blocked by AM251 (CB1 receptor antagonist), but was not influenced by AM630 (CB2 receptor antagonist). In addition, we also observed that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a cell-permeable cAMP analog) reversed the inhibitory effect of CP55940, respectively. In a summary, our observations raise a possibility that CB1R rather than CB2R can downregulate the opening of P2X receptor channels in DH neurons. The reduction of cAMP/PKA signaling is a key element in the inhibitory effect of CB1R on P2X-channel-induced Ca2+ mobilization.

Pax2 is persistently expressed by GABAergic neurons throughout the adult rat dorsal horn

The transcription factor Pax2 is required for the differentiation of GABAergic neurons in the mouse dorsal horn. Pax2 continues to be expressed in the adult murine spinal cord and has been used as a presumed marker of GABAergic neurons in the superficial dorsal horn of the adult mouse, although a strict association between adult Pax2 expression and presence of GABA throughout the dorsal horn has not been firmly established. Moreover, whether Pax2 is selectively expressed in GABAergic dorsal horn neurons also in the rat is unknown. Here, immunofluorescent labeling of Pax2 and GABA in the lumbar spinal cord of adult rats was used to investigate this issue. Indeed, essentially all GABA immunoreactive neurons in laminae I–V were immunolabeled for Pax2. Conversely, essentially all Pax2 immunopositive neurons in these laminae exhibited somatic GABA immunolabeling. These results indicate persistent Pax2 expression in GABAergic neurons in the adult rat dorsal horn, supporting the hypothesis that Pax2 may be required for the maintenance of a GABAergic phenotype in mature inhibitory dorsal horn neurons in the rat. Furthermore, Pax2 may be used as a selective and specific general somatic marker of such neurons.

Noradrenergic inhibition of spinal hyperexcitation elicited by cutaneous cold stimuli in rats with oxaliplatin-induced allodynia: electrophysiological and behavioral assessments.

We investigated the spinal action of noradrenaline on cold-elicited hyperexcitation detected in dorsal horn neurons of rats with allodynia induced by an oxaliplatin (6mg/kg, i.p.) injection. In vivo extracellular recordings from the spinal dorsal horn showed that wide dynamic range neurons responded to cutaneous acetone (10μl) stimulation in normal rats, and cold-elicited firings in oxaliplatin-administered rats were increased with a longer duration, correlated with behavioral responses. These responses were significantly attenuated by spinal administration (50μM) of noradrenaline or its agonists, clonidine (α2), phenylephrine (α1) and isoprenaline (β), in descending order of efficacy. Thus, the inhibitory effect of noradrenaline on spinal oxaliplatin-induced cold hyperexcitation is mediated mainly by activation of α2- and/or α1-adrenoceptors.

Blockade of 5-HT2A receptors at the site of inflammation inhibits activation of spinal dorsal horn neurons in rats.

Repeated inflammation in the periphery is a major cause of chronic inflammatory pain. We have showed that blockade of 5-HT2A receptors in the periphery inhibits repeated inflammation-induced pain hypersensitivity. The present study investigated whether inhibition of 5-HT2A receptor signaling at the site of inflammation could inhibit repeated inflammation-induced neurochemical changes in spinal dorsal horn neurons. Treatment with the 5-HT2A receptor antagonist ketanserin (20μg) in the hindpaw following intraplantar injection of carrageenan inhibited the increase in nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity, a marker of nitric oxide synthase (NOS), in the spinal dorsal horn. Administration of formalin (1%) in the hindpaw following the carrageenan injection evoked a great increase in NADPH-d reactivity in spinal dorsal horn neurons on the ipsilateral and contralateral sides. These changes were abolished by the pretreatment of ketanserin (20μg). The ketanserin treatment also reduced the repeated inflammation-induced expression of protein kinase gamma (PKCγ) in the membrane of spinal dorsal horn neurons and increased PKCγ protein level in the cytosol. Following the treatment with the opioid receptor antagonist naloxone methiodide (5mg/kg, s.c.), the administration of ketanserin failed to inhibit the repeated inflammation-induced increase in NADPH-d reactivity and c-Fos expression in the spinal dorsal horn. These results suggest that 5-HT2A receptor bioactivity in the inflammatory site plays an important role in repeated inflammation-induced central sensitization. The present study supports the notion that targeting 5-HT2A receptors in the periphery can be a promising therapy for relieving chronic inflammatory pain.

Modulating Effect of Peptide Semax on the Level of Synaptic Activity and Short-Term Plasticity in Glutamatergic Synapses of Co-Cultured Dorsal Root Ganglion and Dorsal Horn Neurons of Rats

The effects of a long-term culturing (12 days, in vitro) of the dorsal root ganglion (DRG) and the dorsal horn (DH) neurons with peptide Semax on the level of synaptic activity in co-cultures, as well as short-term plasticity in sensory synapses were studied. It has been revealed that neuronal culturing with peptide at concentrations of 10 and 100 µM leads to increasing the frequency of spontaneous glutamatergic postsynaptic currents in DH neurons by 71.7 ± 1.8% and 93.9 ± 3.1% (n = 6; P < 0.001). Semax does not exert a pronounced effect on the amplitude and frequency of miniature glutamatergic currents, but it causes an increase of the amplitudes of spontaneous postsynaptic currents and an elevation of the quantum content. The data obtained indicate the increase in multivesicular glutamate release efficiency in neural networks of co-cultures following incubation with the peptide. Moreover, Semax (10 and 100 µM) induces changes in the basic parameters of short-term plasticity of sensory synapses, namely, i) an increase in the paired-pulse ratio from (0.53 ± 0.028) (n = 8) to (0.91 ± 0.072) (n = 6, P < 0.01) and (0.95 ± 0.026) (n = 7; P < 0.001); ii) reduced coefficients of variation ratio (CV2/CV1) from (1.49 ± 0.11) (n = 8) to (1.02 ± 0.09) (n = 6; P < 0.05) and (1.11 ± 0.13) (n = 7; P < 0.01) respectively. The results indicate a stimulating effect of Semax on the activity of glutamatergic synapses in neural networks of co-cultures, as well as the ability of the peptide to modulate effectively short-term plasticity of sensory synapses.

Human psychophysics and rodent spinal neurones exhibit peripheral and central mechanisms of inflammatory pain in the UVB and UVB heat rekindling models.

Translational research is key to bridging the gaps between preclinical findings and the patients, and a translational model of inflammatory pain will ideally induce both peripheral and central sensitisation, more effectively mimicking clinical pathophysiology in some chronic inflammatory conditions. We conducted a parallel investigation of two models of inflammatory pain, using ultraviolet B (UVB) irradiation alone and UVB irradiation with heat rekindling. We used rodent electrophysiology and human quantitative sensory testing to characterise nociceptive processing in the peripheral and central nervous systems in both models. In both species, UVB irradiation produces peripheral sensitisation measured as augmented evoked activity of rat dorsal horn neurones and increased perceptual responses of human subjects to mechanical and thermal stimuli. In both species, UVB with heat rekindling produces central sensitisation. UVB irradiation alone and UVB with heat rekindling are translational models of inflammation that produce peripheral and central sensitisation, respectively. The predictive value of laboratory models for human pain processing is crucial for improving translational research. The discrepancy between peripheral and central mechanisms of pain is an important consideration for drug targets, and here we describe two models of inflammatory pain that involve ultraviolet B (UVB) irradiation, which can employ peripheral and central sensitisation to produce mechanical and thermal hyperalgesia in rats and humans. We use electrophysiology in rats to measure the mechanically- and thermally-evoked activity of rat spinal neurones and quantitative sensory testing to assess human psychophysical responses to mechanical and thermal stimulation in a model of UVB irradiation and in a model of UVB irradiation with heat rekindling. Our results demonstrate peripheral sensitisation in both species driven by UVB irradiation, with a clear mechanical and thermal hypersensitivity of rat dorsal horn neurones and enhanced perceptual responses of human subjects to both mechanical and thermal stimulation. Additional heat rekindling produces markers of central sensitisation in both species, including enhanced receptive field sizes. Importantly, we also showed a correlation in the evoked activity of rat spinal neurones to human thermal pain thresholds. The parallel results in rats and humans validate the translational use of both models and the potential for such models for preclinical assessment of prospective analgesics in inflammatory pain states.

Gabapentin prevents oxaliplatin-induced central sensitization in the dorsal horn neurons in rats.

The present study aims to study the alteration of glutamatergic transmission in the dorsal horn neurons and the effect of gabapentin on oxaliplatin-induced neuropathic pain in rats. Oxaliplatin (5 mg/kg) or saline was administered to adult male Sprague-Dawley rats. Gabapentin (60 mg/kg, IP) or vehicle was injected daily. Mechanical allodynia was assessed using a series of von Frey filaments. The expression of glutamate receptor subunits (NR2B and GluR1) and brain-derived neurotrophic factor (BDNF) was measured in the dorsal horn. The glutamatergic strength was recorded in the spinal cord slices. Administration of oxaliplatin induced significant hyperreactivity to mechanical stimuli in rats, which was attenuated by gabapentin. Significant increase in the expression of BDNF was found in the dorsal horn in rats receiving oxaliplatin, which was prevented by gabapentin. Further studies also observed a significant increase in the expression of GluR1 and NR2B, as well as enhanced glutamatergic transmission in the dorsal horn neurons in rats treated with oxaliplatin. The upregulation of glutamatergic transmission was significantly reversed by gabapentin. These results illustrated an increased expression of BDNF and enhanced glutamatergic transmission in rats with oxaliplatin-induced neuropathic pain, which was markedly attenuated by gabapentin.

Inflammation of the thoracolumbar fascia excites and sensitizes rat dorsal horn neurons.

Recent data show that the thoracolumbar fascia can be a source of pain. However, the spinal neuronal mechanisms underlying pain from a pathologically altered fascia are unknown. The present study aimed at finding out how dorsal horn neurons react to input from a chronically inflamed thoracolumbar fascia. Recordings from rat dorsal horn neurons were made in the spinal segment L3. Twelve days before the recordings, the thoracolumbar fascia was inflamed by injection of complete Freund's adjuvant. Control animals received an injection of isotonic saline. In addition, behavioural experiments were carried out. Neurons in the spinal segment L3 do not normally receive input from the fascia, but 11.1% of the neurons did when the fascia was inflamed. Compared with control, the proportion of neurons having input from all deep somatic tissues rose from 10.8% to 33.3% (p < 0.02). Moreover, many neurons acquired new deep receptive fields, most of which were located in the hindlimb (p < 0.04). Surprisingly, the pressure pain threshold of the inflamed rats did not change, but they showed a reduction in exploratory activity. One of the prominent findings was the appearance of new receptive fields in deep tissues of the hindlimb. Together with the expansion of the spinal target region of fascia afferents into the segment L3, the appearance of new receptive fields is a possible explanation for the spread of pain in patients with non-specific low back pain.

Immobilization stress sensitizes rat dorsal horn neurons having input from the low back.

Stress is known to promote several forms of muscle pain including non-specific low back pain. However, the question if stress alone activates nociceptive central neurons has not been studied systematically. Here, we investigated the influence of repeated immobilization stress on dorsal horn neurons and behaviour in the rat. The stress consisted of immobilization in a narrow tube for 1 h on 12 days. Single dorsal horn neurons were recorded with microelectrodes introduced into the spinal segment L2. In this segment, about 14% of the neurons responded to mechanical stimulation of the subcutaneous soft tissues of the low back in naïve rats. The neurons often behaved like wide dynamic range cells in that they had a low mechanical threshold and showed graded responses to noxious stimuli. The stress-induced changes in neuronal response behaviour were (1) appearance of new receptive fields in the deep tissues of the hindlimb, (2) increased input from deep soft tissues, but unchanged input from the skin and (3) significant increase in resting activity. Surprisingly, the pressure-pain threshold of the low back remained unchanged, although dorsal horn neurons were sensitized. In the open field test, the rats showed signs of increased anxiety. This study shows that stress alone is sufficient to sensitize dorsal horn neurons. The data may explain the enhanced pain low back patients report when they are under stress. The increased resting discharge may lead to spontaneous pain.

Effect of intrathecal resveratrol on activation of CaMKII in spinal dorsal horn neurons of rats with bone cancer pain

Objective To evaluate the effect of intrathecal resveratrol on activation of Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) in spinal dorsal horn neurons of rats with bone cancer pain. Methods Thirty-two adult female Sprague-Dawley rats, weighing 180-220 g, were equally randomized into 4 groups using a random number table: sham operation group (group S), bone cancer pain group (B group), and bone cancer pain + solvent control group (BD group). Walker 256 mammary gland cancer cell suspension (4×105 cells/ml) 5 μl was injected into the medullary cavity of the right tibia in B, BR and BD groups.Normal saline 5 μl was given in group S. On 12, 13 and 14 days after injection of mammary gland cancer cells, resveratrol 200 μg/10 μl was injected intrathecally once a day in group BR, and 1% dimethyl sulfoxide 10 μl was intrathecally injected once a day in group BD.Before injection of mammary gland cancer cells (T0) and on 3, 5, 7, 10, 12 and 14 days after injection of mammary gland cancer cells (T1-6), mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured.The rats were then sacrificed and L4, 5 segments of the spinal cord were removed for confirmation of the location of phosphorylated CaMKⅡ (p-CaMKⅡ) in spinal dorsal horn neurons (by immunofluorescence) and for detection of p-CaMKⅡ expression (using Western blot). Results Compared with group S, MWT and TWL were significantly decreased at T2-6, and p-CaMKⅡ expression was up-regulated at T6, and p-CaMKⅡ was mainly co-expressed with neurons in B, BR and BD groups.Compared with group B, MWT and TWL were significantly increased at T5, 6, and p-CaMKⅡ expression was down-regulated at T6 in group BR.There was no significant difference in MWT, TWL, and p-CaMKⅡ expression at each time point between group B and group BD. Conclusion Resveratrol can alleviate hyperalgesia in rats with bone cancer pain and inhibited activation of CaMKⅡ in spinal dorsal horn neurons may be involved in the mechanism. Key words: Phenols; Injections, spinal; Bone neoplasms; Pain; Spinal cord; Neurons; Calcium-calmodulin-dependent protein kinase type 2

Strong Manual Acupuncture Manipulation Could Better Inhibit Spike Frequency of the Dorsal Horn Neurons in Rats with Acute Visceral Nociception.

Afferent impulses from visceral nociception can be regulated by acupuncture at spinal cord level; however, the effects of different manual acupuncture (MA) manipulations on the afferent impulses are still unknown. Here, we analyzed the spike frequency of excitatory gastric-related wide dynamic range (WDR) neurons in spinal dorsal horn (SDH) following acute gastric distension (GD) in rats and compared their responses to MA manipulations with four different frequencies (0.5, 1, 2, and 3 Hz) at Zusanli (ST36). Results indicated that the spike frequency was increased by acute GD stimulation. Under acute GD circumstances, the spike frequency was further activated by weak MA stimulation (0.5 and 1 Hz), while being significantly inhibited by strong MA stimulation (2 and 3 Hz). After 10 minutes of the strong MA stimulation, same intensity of acute GD caused less spike frequency. Our previous researches had demonstrated that different MA manipulations could increase spike frequency in an intensity-dependent manner in normal rats; these findings suggest that acupuncture may have different modulatory effects depending on the state of the stomach. Since neuronal spike frequency was related to the level of nociception, the results suggest that strong MA manipulation may have better effect on acute visceral nociception.