Attenuation of mechanical but not thermal hyperalgesia by electroacupuncture with the involvement of opioids in rat model of chronic inflammatory pain

Opioid peptides have been proven effective in reducing the sign of hyperalgesia associated with inflammation. Electroacupuncture (EA) produces antinociception via release of endogenous opioid peptides in normal rats. Moreover, intrathecal injection of dynorphin has antinociceptive effect in rats. The present study was designed to examine whether EA has effect on the thermal and mechanical hyperalgesia in rat model of complete Freund's adjuvant (CFA)-induced inflammatory pain. The results are the following: (1) single session of 100Hz EA (0.5-1.0-1.5 mA, 10 min for each intensity) at both Zusanli (ST 36) and Sanyinjiao acupoints (SP 6) significantly increased mechanical withdrawal threshold determined by von Frey filaments but not with thermal withdrawal latency that is determined by hot plate (52 +/- 0.2 degrees C); (2) 100 Hz EA applied twice a week for 4 weeks and showed a significant decrease in the mechanical hyperalgesia at the third and fourth week, with no effect on thermal hyperalgesia; (3) naloxone (20 mg kg(-1)) had the ability to reverse the inhibition of the mechanical hyperalgesia produced by a single session of EA. In conclusion, the present results indicate that a single or repetitive EA could reduce mechanical hyperalgesia, but not thermal hyperalgesia, in CFA-inflammatory pain rats, and the opioid system might be involved in these effects.     

Spinal inhibition of p38 MAP kinase reduces inflammatory and neuropathic pain in male but not female mice: Sex-dependent microglial signaling in the spinal cord

Previous studies have shown that activation of p38 mitogen-activating kinase (MAPK) in spinal microglia participates in the generation of inflammatory and neuropathic pain in various rodent models. However, these studies focused on male mice to avoid confounding effects of the estrous cycle of females. Recent studies have shown that some spinal pro-inflammatory signaling such as Toll-like receptor 4-mediated signaling contributes to pain hypersensitivity only in male mice. In this study we investigated the distinct role of spinal p38 in inflammatory and neuropathic pain using a highly selective p38 inhibitor skepinone. Intrathecal injection of skepinone prevented formalin induced inflammatory pain in male but not female mice. Furthermore, intrathecal skepinone reduced chronic constriction injury (CCI) induced neuropathic pain (mechanical allodynia) in male mice on CCI-day 7 but not CCI-day 21. This male-dependent inhibition of neuropathic pain also occurred in rats following intrathecal skepinone. Nerve injury induced spinal p38 activation (phosphorylation) in CX3CR1-GFP⁺ microglia on CCI-day 7, and this activation was more prominent in male mice. In contrast, CCI induced comparable microgliosis and expression of the microglial markers CX3CR1 and IBA-1 in both sexes. Notably, intraperitoneal or local perineural administration of skepinone inhibited CCI-induced mechanical allodynia in both sexes of mice. Finally, skepinone only reduced the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in lamina IIo neurons of spinal cord slices of males 7 days post CCI. Therefore, the sex-specific p38 activation and signaling is confined to the spinal cord in inflammatory and neuropathic pain conditions.     

Interferon-γ and lipopolysaccharide reduce cAMP responses in cultured glial cells: Reversal by a type IV phosphodiesterase inhibitor

The aim of the present study was to determine whether two classical macrophage activators, bacterial lipopolysaccharide (LPS) and interferon-γ (IFN-γ) could affect the accumulation of the second messenger cAMP in cultured rat microglia and astrocytes. Purified microglia and astrocyte secondary cultures obtained from the neonatal rat were grown for 3 days in basal medium Eagle (BME) + 10% fetal calf serum (FCS). Exposure of microglia to LPS resulted into a dose- and time-dependent decrease in the accumulation of cAMP induced by receptor-mediated (isoproterenol or prostaglandin E₂) or direct (forskolin) activation of adenylate cyclase. The inhibitory effect of LPS was rapid (a 10 min preincubation was sufficient to approach a maximal effect), occurred at low doses (IC₅₀ = 1.2 ng/ml), and was not abrogated by pertussis toxin. A selective inhibitor of type IV phosphodiesterase (rolipram, 100 nM) prevented the effect of LPS on cAMP accumulation, while inhibitors of other forms of phosphodiesterase were unable to do so. IFN-γ (100 u/ml) also caused a depression of the evoked cAMP accumulation in microglia after a 10 min preincubation, and its effect was prevented by rolipram, as in the case of LPS. Astrocytes differed from microglia in that LPS (1–100 ng/ml) did not inhibit the accumulation of cAMP induced by either isoproterenol or forskolin; on the other hand, IFN-γ did have an inhibitory effect (though less pronounced than in microglia) that could be prevented by rolipram. Our observations indicate that two potent activators of microglia acting at different receptors, LPS and IFN-γ, can diminish the accumulation of cAMP through a common mechanism, the stimulation of a specific form of cAMP phosphodiesterase. The fact that IFN-γ, but not LPS, was effective in astrocytes suggests that LPS receptors are scarcely, if at all, expressed in these cells, or that they are differently coupled to second messengers. Selective inhibitors of type IV phosphodiesterase might prevent some of the obnoxious actions of LPS or IFN-γ in the living organism. © 1995 Wiley-Liss, Inc.     

Nerve injury-induced mechanical but not thermal hyperalgesia is attenuated in neurokinin-1 receptor knockout mice

Mice lacking the gene encoding for substance P and neurokinin A, or the NK-1 receptor, exhibit alterations in behavior to various acute nociceptive stimuli. However, behavioral responses of NK-1 mutant animals have not been well characterized in models of chronic pain. We studied the behavioral responses of NK-1 knockout and wild-type control mice to thermal and mechanical stimuli before and after inducing chronic neuropathic pain by unilateral ligation of the L5 spinal nerve. Mechanical hyperalgesia was evaluated by determining the frequency of withdrawal to von Frey monofilaments applied to the hind paws. Nerve injury-induced hyperalgesia to thermal stimuli was examined by determining responses to radiant heat and cooling stimuli. The contribution of the sympathetic nervous system to mechanical hyperalgesia was evaluated by administering 3 mg/kg phentolamine, an alpha-adrenergic antagonist, subcutaneously. Following spinal nerve injury, withdrawal frequencies to mechanical stimulation increased in wild-type mice within 1 day and persisted during the 9-week observation period, whereas in the knockout mice, withdrawal frequencies did not increase significantly. In contrast, withdrawal latencies to radiant heat decreased up to 2 weeks after nerve injury in both the NK-1 and the wild-type mice. Similarly, the increase in withdrawal frequency to the cooling stimuli following the nerve injury was not different in the NK-1 knockout and wild-type mice. Mechanical hyperalgesia in the wild-type mice was not reversed by systemic administration of phentolamine, suggesting that the pain is not sympathetically maintained. The results indicate that NK-1 receptors contribute to the development of mechanical, but not thermal, hyperalgesia in neuropathic pain.     

Excitotoxic lesioning of the rat basal forebrain with S-AMPA: consequent mineralization and associated glial response

Regional depositions of calcium within the basal ganglia, cortex, cerebellum, and white matter and at perivascular sites have been observed in several pathological conditions. These generally indicate signs of ongoing apoptosis or necrotic processes, whereby the activation of glutamate receptors causes a rise in intracellular calcium levels leading to mineralization of neurons, and ultimately to cell death. The selective degeneration of cholinergic neurons in the basal forebrain is a major neuropathological component of Alzheimer's disease, and may result in abnormal deposition of calcium. In experimental models, selective lesions of the basal forebrain can be induced by intraparenchymal infusions of excito- or immunotoxins targeting cholinergic neurons. Excitotoxic lesions are often accompanied by calcium deposition within affected areas. In a previous study we also noted the presence of unusual deposition in areas close to the site of injections following unilateral S-AMPA-induced lesions of the basal forebrain (T. Perry, H. Hodges, and J. A. Gray, 2001, Brain Res. Bull. 54, 29-48). In this paper, we have characterized these deposits histologically and evaluated the microglial (CD11b) and astrocytic (GFAP) responses at 8 and 16 weeks following lesioning of the nucleus basalis magnocellularis with S-AMPA. The resulting deposits were heterogeneous in morphology and composed primarily of calcium. Small granular deposits were detected around blood vessels, whereas larger calcospherites were situated within the parenchyma. These deposits were more widely dispersed at 16 weeks postlesioning, affected neighboring nuclei, and displayed a progressive increase in size and frequency of occurrence. However, calcification within these regions was differentially associated with microglial and astrocytic reactivity at the two time points. Both microglial and astrocytic responses were pronounced at 8 weeks, whereas at 16 weeks, astrocytic reactivity prevailed and the microglial response was markedly attenuated. Importantly, the pattern of reactivity for microglia detected at 8 weeks was specifically localized to vulnerable nucleated areas prior to their substantial accumulation of calcium deposits, which was clearly evident by 16 weeks. We suggest that the initial microglial response could be used as a selective predictor of tissue necrosis and subsequent calcification, and that astrocytes, which form a glial scar in the affected tissues, may contribute toward the buildup of calcium deposits. The functional relevance of these findings is discussed.     

Methylprednisolone inhibits early inflammatory processes but not ischemic cell death after experimental spinal cord lesion in the rat

Experimental studies and clinical observations show that spinal cord lesions are greatly enlarged by a process called secondary cell death. A detailed understanding of the molecular and cellular processes underlying these events is still lacking. In clinical studies using methylprednisolone in spinal cord injured patients a mega-dose of methylprednisolone applied during the first dew hours after injury was found to improve the neurological outcome. In the present study the possible neuroprotective mechanism of methylprednisolone was assessed by histologically studying its effect on the extent of secondary cell death and on early inflammatory reactions following partial transection of the spinal cord in the rat. Our results show that a single high dose of 30 or 60 mg/kg methylprednisolone affects neither the time course nor the extent of secondary cell death. In contrast, methylprednisolone markedly suppressed the invasion of the injured spinal cord tissue by polymorphonuclear granulocytes and macrophages. The role of these inflammatory cells in traumatic CNS lesions is very unclear at present. It is possible that they lead to further damage of the injured spinal cord tissue and that the beneficial effect of methylprednisolone is at least partially due to its anti-inflammatory effect, thereby inhibiting bystander damage of invading inflammatory cells.     

Analgesic action of suspended moxibustion in rats with chronic visceral hyperalgesia correlates with enkephalins in the spinal cord

Rats that modeled chronic visceral hyperalgesia received suspended moxibustion at bilateral Tianshu (ST25) and Shangjuxu (ST37) once daily over a period of 7 days. Results show that suspended moxibustion significantly depressed abdominal withdrawal reflex scores and increased enkephalin concentration in the spinal cord. The experimental findings suggest that spinal enkephalins contributed to the analgesic effect of suspended moxibustion in rats with chronic visceral hyperalgesia.     

Evidence for suppression of electroacupuncture on spinal glial activation and behavioral hypersensitivity in a rat model of monoarthritis

Our previous study demonstrated that single intrathecal (i.t.) application of fluorocitrate, a glial metabolic inhibitor, synergized electroacupuncture (EA) antagonizing behavioral hypersensitivity in complete Freund's adjuvant (CFA)-induced monoarthritic rat. To further investigate the relationship between spinal glial activation and EA analgesia, the present study examined the effects of multiple EA on spinal glial activation evoked by monoarthritis (MA). The results showed that (1) unilateral intra-articular injection of CFA produced a robust glial activation on the spinal cord, which was associated with the development and maintenance of behavioral hypersensitivity; (2) multiple EA stimulation of ipsilateral "Huantiao" (GB30) and "Yanglingquan" (GB34) acupoints or i.t. injection of fluorocitrate (1 nmol) significantly suppressed spinal glial activation; (3) inhibitory effects of EA on spinal glial activation and behavioral hypersensitivity were significantly enhanced when EA combined with fluorocitrate, indicating that disruption of glial function may potentiate EA analgesia in inflammatory pain states. These data suggested that analgesic effects of EA might be associated with its counter-regulation to spinal glial activation, and thereby provide a potential strategy for the treatment of arthritis.     

福尔马林炎症痛诱导的大鼠脊髓后角一氧化氮合酶的变化

用ＮＡＤＰＨ－黄递酶（ＮＡＤＰＨ－d）组化法观察福尔马林炎症性痛及痛过敏过程中脊髓后角一氧化氮合酶（NOS)的变化,特别是其时间特征。结果显示：与对照组相比,注射福尔马林后１２、２４及４８h组脊髓后角ＮＡＤＰＨ－d阳性细胞的数目及染色深度均显著增加,但以２４h组增加最为明显。结果表明福尔马林炎症性痛及痛过敏过程中,脊髓后角ＮＯＳ活性增强,且这种增强有一定的时间变化特征。     

Long-term microglial and astroglial activation in the hippocampus of trimethyltin-intoxicated rat: stimulation of NGF and TrkA immunoreactivities in astroglia but not in microglia

In the present study we investigated the microglial and astroglial response after trimethyltin (TMT) exposure over a prolonged period of time. Male Wistar rats were given a single dose of TMT (8 mg/kg, i.p.) and survived 4, 7, 21, 60 and 180 days after the administration of the toxin. Histochemistry (Griffonia simplicifolia lectin staining) and immunocytochemistry for GFAP were applied to identify micro- and astroglial cells, respectively. To assess the trophic response of glial cells (NGF and TrkA expression), single or double staining experiments were performed. In addition, the biochemical evaluation of GFAP and NGF were carried out at chosen timepoints using immunoblotting technique and ELISA, respectively. The main findings of our study were as follows. (1) A protracted activation of microglia (at least up to 2 months posttreatment). (2) A long-lasting expression of GFAP immunoreactivity (at least up to 6 months posttreatment) and a steady increase in GFAP content (at least up to 2 months posttreatment). (3) The appearance of enormously enlarged, round-shape astrocytes exclusively localized to CA1 and observed 2 months posttreatment. (4) The stimulation of NGF and TrkA expression in reactive astrocytes. (5) The strongest activation of micro- and astroglia coincided with the most prominent neurodegeneration in the hippocampus, i.e., in CA4/CA3c and CA1. It is tempting to assume that the activation of glial cells in the hippocampal areas particularly vulnerable to TMT may affect neuronal fate after neurotoxic insult.     

Acute dispersion of glial cells following transplantation into the myelin-deficient rat spinal cord

Evaluation of glial cell migration following transplantation can be difficult as the force of the injection itself may cause the cells to become immediately dispersed. In this study we evaluated the extent of spread of cells after injection of 1 μl of a dissociated cell suspension (50,000 cells/μl) into the dorsal columns of the thoracolumbar spinal cord in the neonatal myelin-deficient (md) rat. Spinal cords were examined at 0, 4, and 24 h after injection to determine the dispersion of cells away from the initial site of deposition. Examination of skip-serial sections collected at 50-μm intervals rostral and caudal to the site of transplantation showed that the injection could result in a spread of transplanted cells up to 1,600 μm. Migration should therefore be defined as the detection of cells beyond the rostral-caudal boundaries defined by the injection deposition. Cell dispersion should be taken into account when evaluating the results of migration in previous and future experiments concerning glial cell transplantation. © 1995 Wiley-Liss, Inc.     

Endothelial cell loss is not a major cause of neuronal and glial cell death following contusion injury of the spinal cord

Contusion of the spinal cord causes an immediate local loss of neurons and disruption of vasculature; additional loss continues thereafter. To explore the possibility of a causal link between delayed endothelial cell (EC) death and secondary neural cell loss, we evaluated neural and endothelial cell survival, and measured inflammatory cell infiltration, at times up to 48 h after contusion injury to the adult rat thoracic spinal cord. Female Fischer rats (200 g), subjected to moderate (10 g x 12.5 mm) weight drop injuries by the MASCIS (NYU) impactor, were analyzed at 15 min and at 1, 8, 24 and 48 h. ECs, neurons, astrocytes, oligodendrocytes, neutrophils and activated macrophages/microglia were counted in transverse sections. At the injury site, 90% of all neurons died within 48 h of injury; no medium-large diameter neurons survived beyond 48 h. EC death occurred with kinetics similar to glial cell death. Because, in the injury site, most cell death occurred before 8 h, it preceded inflammatory cell infiltration. Three millimeters rostral and caudal to the injury epicenter neuronal numbers were stable for 8 h, and a sharp decrease in neuronal numbers beginning at 8 h strongly correlated with the onset of inflammatory cell infiltration. Glial and blood vessel numbers remained relatively stable in these areas up to 48 h. These results suggest that the loss of ECs during the first 48 h after a contusion injury is not a major cause of neuronal and glial cell death and, in tissue adjacent to the epicenter, inflammatory cell infiltration leads to neuronal loss.     

Long-term effects of spinal cord stimulation in chronic pain syndromes

Summary A total of 50 patients with chronic pain syndromes were selected for treatment with spinal cord stimulation. Correct positioning of electrodes was obtained in 44 patients, leading to an initial alleviation of pain in 25 patients. In 6 patients, electrodes (though still effective in 4) had to be removed because of surgical complications within the first 5 months of use. Only 8 patients had at least some beneficial effect lasting for more than 3 years. The long-term results in patients with more severe psychological disturbances were no worse than those of the other patients.     

Nitric oxide-dependent long-term potentiation revealed by real-time imaging of nitric oxide production and neuronal excitation in the dorsal horn of rat spinal cord slices

Nitric oxide (NO) is thought to be involved in the central mechanism of hyperalgesia and allodynia at the spinal level. Recently, we reported that NO played an important role in the induction of long-term potentiation (LTP) of synaptic strength in spinal dorsal horn, which is believed to underlie hyperalgesia and allodynia. In this study, to elucidate the relationship of NO to LTP in spinal dorsal horn, we measured the spatiotemporal distribution of NO signal with the NO-sensitive dye, DAR-4M, and neuronal excitation with the voltage-sensitive dye, RH482, in rat spinal cord slices, elicited by dorsal root stimulation. In superficial dorsal horn, neuronal excitation evoked by C fiber-activating dorsal root stimulation was potentiated for more than 2 h after low-frequency conditioning stimulation (LFS, 240 pulses at 2 Hz for 2 min). In the same slices that exhibited LTP, NO was produced and distributed in the superficial dorsal horn during the delivery of LFS, and the amplitude of LTP and amount of NO production showed close correlation from slice to slice. LTP and production of NO were inhibited in the presence of the NO synthase inhibitors and an inhibitor of heme oxygenase, the synthetic enzyme for carbon monoxide (CO). These results suggest that production and distribution of NO is necessary for the induction of LTP in spinal dorsal horn, and that CO contributes to the LTP induction and NO production by LFS.     

Contribution of capsaicin-sensitive primary afferents to mechanical hyperalgesia induced by ventral root transection in rats: the possible role of BDNF

Objective: A recent study showed that brain-derived neurotrophic factor (BDNF) may play a role in the development of the neuropathic pain resulting from injury to motor efferent fibres, such as that in the ventral root transection (VRT) model. Capsaicin stimulation of afferent fibres was also shown to result in the release of BDNF into the spinal cord. Here, the effects of ablation of capsaicin-sensitive primary afferents (CSPAs) by local application of capsaicin on the sciatic nerve on VRT-induced mechanical hyperalgesia were observed.

Methods: The paw withdrawal mechanical threshold (PWMT) was measured before and then 1 and 3 days and 1, 2, 3, 4 and 6 weeks after VRT.

Results: The results showed that local application of capsaicin significantly inhibited the decrease in the PWMT induced by VRT, suggesting the inhibitory effect of locally delivered capsaicin. Furthermore, intrathecal administration of exogenous BDNF not only produced mechanical hyperalgesia but also significantly blocked the inhibitory effect of capsaicin.

Conclusion: Taken together, the results of this study suggest that CSPA fibres may contribute to mechanical hyperalgesia in the VRT model.     

Neuronal nitric oxide synthase (nNOS) mRNA is down-regulated, and constitutive NOS enzymatic activity decreased, in thoracic dorsal root ganglia and spinal cord of the rat by a substance P N-terminal metabolite

Nitric oxide (NO) in the spinal cord plays a role in sensory and autonomic activity. Pain induced by acetic acid in the abdominal stretch (writhing) assay and hyperalgesia associated with chronic pain are highly sensitive to NO synthase (NOS) inhibitors. Because substance P (SP) is released and up-regulated in some models of chronic pain, we hypothesized that an accumulation of SP metabolites may influence NOS expression and activity. To test this hypothesis, we examined the effect of intrathecally (i.t.) injected substance P (1-7) [SP(1-7)], the major metabolite of SP in the rat, on neuronal NOS (nNOS) mRNA in the thoracic and lumbar spinal cord, dorsal root ganglia (DRG) and on the corresponding constitutive NOS (cNOS) enzyme activity. Detected using quantitative RT-PCR, nNOS mRNA content in the thoracic spinal cord was decreased 6 h after injection of 5 micromol of SP(1-7) and returned to control 2 days later. In thoracic DRG, nNOS mRNA was reduced 48 h after SP(1-7). The cNOS enzymatic activity in thoracic spinal tissue was gradually decreased to a minimum at 72 h. Down-regulation of NOS by SP(1-7) in the thoracic area appears to be highly associated with capsaicin-sensitive primary afferent neurons. No similar changes in either parameter were measured in the lumbar area after SP(1-7). These data suggest that N-terminal SP fragments, which are known to cause long-term antinociception in the writhing assay, may do so by their ability to down-regulate NO synthesis along nociceptive pathways.     

Distribution and differentiation of A2B5+ glial precursors in the developing rat spinal cord

In many regions of the rat central nervous system, oligodendrocytes develop from migratory A2B5⁺ precursor cells. In the rat spinal cord, during early embryonic development the capacity for oligodendrogenesis appears to be restricted to ventral regions of the spinal cord, while cultures of postnatal rat spinal cord contain a distinct population of A2B5⁺ astrocyte precursors. To determine if, as in other regions of the CNS, spinal cord A2B5⁺ cells give rise directly to oligodendrocytes and astrocytes, the initial distribution, and subsequent dispersion, proliferation, and differentiation of spinal cord A2B5⁺ cells have been examined in both explant and dissociated cell cultures. Spinal cord oligodendrocytes develop from A2B5⁺ cells. At E14, A2B5⁺ cells are restricted to ventral regions of the spinal cord and as development proceeds they become more uniformly distributed throughout the spinal cord. In explant cultures, greater than 95% of the explants that contain oligodendrocytes also contain A2B5⁺ cells and a proportion of mature oligodendrocytes retain detectable A2B5 immunoreactivity briefly on their surface. The maturation of spinal cord oligodendrocyte precursors occurs in a number of distinct stages characterized by the expression of O4 immunoreactivity, which first appears at E16, and GC immunoreactivity, which first appears at E18. As spinal cord oligodendrocyte precursors acquire O4 immunoreactivity they appear to lose the ability to proliferate in response to PDGF but retain the ability to proliferate in response to bFGF, suggesting that the control of proliferation of oligodendrocyte precursors is, in part, dependent on their maturational state. In the presence of high serum, spinal cord A2B5⁺ cells fail to develop in isolated E14 dorsal spinal cord cultures, while in ventral cultures they subsequently differentiate into A2B5⁺ astrocytes suggesting that A2B5⁺ astrocyte precursors are also initially ventrally located. Unlike oligodendrocyte differentiation, however, the differentiation of spinal cord A2B5⁺ cells into astrocytes is delayed in early embryonic-derived cultures compared to those from older animals. These observations suggest that local influences may regulate the timing of spinal cord A2B5⁺ astrocyte development, but not spinal cord oligodendrocyte development. © 1994 Wiley-Liss, Inc.