Use of [125I]deoxycytidine to detect herpes simplex virus-specific thymidine kinase in tissues of latently infected guinea pigs

The footpad skin and the lumbosacral dorsal root ganglia were removed from inbred guinea pigs at different times after subcutaneous infection with herpes simplex virus type 2 (HSV-2) in both hind footpads. These tissues, shown by our previous study to harbor latent HSV, were dispersed into single cells. The presence of virus-specific thymidine kinase (TK) in these cells was assayed by the uptake and phosphorylation of [125I]deoxycytidine in culture. [125I]deoxycytidine was shown to be a specific substrate for the HSV-coded TK. The method could detect herpes TK activity in a culture of 10(6) cells with less than 0.1% of the cells being virally infected. The enzyme was readily detected in footpad cells of acutely (24 h) but not of latently (14 days to 1 year) infected guinea pigs. No herpes TK was found either in the sensory ganglionic cells of guinea pigs during the early and late phases of latent infection. It is concluded that HSV-2, while residing in the footpads and the lumbosacral ganglia of the guinea pig during latent infection, does not express any viral TK function.     

C-fos antisense oligodeoxynucleotide decreases subcutaneous bee venom injection-induced nociceptive behavior and fos expression in the rat

Oligodeoxynucleotide complementary to c-fos mRNA was applied to characterize its effect on the spinal cord Fos expression and relevant nociceptive behaviors challenged by subcutaneous injection of bee venom to the rat hind paw. Nociceptive behavioral responses (spontaneous pain and hyperalgesia) following bee venom (0.2 mg/50 microl) injection were assessed in adult male Sprague-Dawley rats receiving intrathecal administration of c-fos antisense oligodeoxynucleotide (ASO, 50 microg/10 microl), sense oligodeoxynucleotide (SO, 50 microg/10 microl) and saline (10 microl) 4 h prior to bee venom injection. The lumbar spinal cord expression of Fos protein 2 h after bee venom injection in the ASO-, SO- and saline-treated animals was observed by immunohistochemistry. The results showed that pretreatment of c-fos ASO markedly reduced the flinching response and primary thermal hyperalgesia, but without significant effects on mechanical hyperalgesia and secondary thermal hyperalgesia. At the same time, ASO treatment also significantly decreased the expression of Fos protein within the lumbar region of the spinal cord ipsilateral to the injection. The results provide further evidence that Fos protein contributes to the activation of the spinal dorsal horn neurons and the generation and/or maintenance of spontaneous pain and primary thermal hyperalgesia induced by subcutaneous injection of bee venom.     

A fluorescence-immunohistochemical study on phosphorylation of ERK1/2, p38, and STAT3 in rat dorsal root ganglia following noxious stimulation of hind paw sensory neurons

A fluorescence-immunohistochemical investigation was performed in lumbar dorsal root ganglia (DRGs) neurons of the rat with regard to ERK1/2-, p38- and STAT3-phosphorylation in response to nociceptor activation in the rat. The stimuli applied were perineural capsaicin treatment of the sciatic nerve, mustard oil application to the hind paw and heat or cold stimulation of the hind paw. The time points of investigations were 15 min/30 min after perineural capsaicin, 30 min/2 h/4 h for mustard oil, 10 min/4 h for cold and 30 min/2 h/8 h for the heat stimulus. All four stimuli lead to a time-dependent, significant 2-3 fold increase in the number of small and medium size DRG cells displaying cytoplasmic staining for p-ERK1/2, but to no activation of satellite cells. Phosphorylated p38 immunoreactivity was increased in the cytoplasma of DRG cells at 2 h after the mustard oil treatment of the hind paw and 30 min after the perineural capsaicin application to the sciatic nerve axons, but not following heat or cold stimuli to the hind paws. Phospho-STAT3 staining was characteristically observed as nuclear and cytoplasmic staining. It was found increased after the perineural capsaicin application to the sciatic nerve axons, however, no marked increase was found with the other 3 noxious stimuli. The present results show that sensory neurons respond with a selective long-lasting increase in p-ERK1/2 in small and medium-size DRG cells, when their axons or axon terminals are stimulated by capsaicin, mustard oil, noxious heat or noxious cold.     

Hyperexcitable neurons and altered non-neuronal cells in the compressed spinal ganglion

The cell body or soma in the dosal root ganglion (DRG) is normally excitable and this excitability can increase and persist after an injury of peripheral sensory neurons. In a rat model of radicular pain, an intraforaminal implantation of a rod that chronically compressed the lumbar DRG ("CCD" model) resulted in neuronal somal hyperexcitability and spontaneous activity that was accom-panied by hyperalgesia in the ipsilateral hind paw. By the 5th day after onset of CCD, there was a novel upregulation in neuronal expression of the chemokine, monocyte chemoattractant protein-1 (MCP- 1 or CCL2) and also its receptor, CCR2. The neurons developed, in response to topically applied MCP-1, an excitatory response that they normally do not have. CCD also activated non-neuronal cells including, for example, the endothelial cells as evidenced by angiogenesis in the form of an increased number of capillaries in the DRG after 7 days. A working hypothesis is that the CCD induced changes in neurons and non-neuronal cells that may act together to promote the survival of the injured tissue. The release of ligands such as CCL2, in addition to possibly activating nociceptive neurons (maintaining the pain), may also act to preserve injured cells in the face of ischemia and hypoxia, for example, by promoting angiogenesis. Thus, somal hyperexcitability, as often said of inflammation, may represent a double edged sword.     

The antinociceptive effect of tramadol on a model of neuropathic pain in rats

The antinociceptive activity of tramadol was investigated on the vocalization threshold to paw pressure in a rat model of unilateral mononeuropathy produced by loose ligatures around the common sciatic nerve. Despite the analgesic activity of tramadol was clearly established in motor and sensory responses of the nociceptive system in rats, the effect of this atypical opioid on experimental neuropathic pain models is not investigated. The intraperitoneally injected tramadol (2.5, 5 and 10 mg/kg) produced a potent and dose-dependent antinociceptive effect on both lesioned and non-lesioned hind paws. However, the analgesic effect on the lesioned paw was significantly more potent than the non-lesioned paw. This effect was partially antagonized by intraperitoneally administered naloxone (0.1 mg/kg) suggesting an additional non-opioid mechanism. Our results suggest that tramadol may be useful for the alleviation of some symptoms in peripheral neuropathic conditions     

Effect of Brand's glucosamine with essence of chicken on collagen-induced arthritis in rats

The anti-arthritic effects of glucosamine incorporated in a chicken-meat extract known as Brand's Glucosamine with Essence of Chicken versus glucosamine or Essence of Chicken (EOC) alone were investigated on collagen induced arthritis (CIA) in dark agouti (DA) rats. Four groups of rats received basic food (control), 1.2% glucosamine (GLU), 0.8% EOC and 1.2% GLU + 0.8% EOC (GLU + EOC) admixed with basic food for 25 days following CIA. Foot pads were isolated on day 25 for histopathological evaluation. Clinical assessment of hind paw swelling as measured by foot pad volumes and histopathological scoring based on the degree of edema, periosteal new bone formation, periostitis and inflammatory cell infiltration of the isolated foot pad were performed. Arthritic rats given GLU + EOC showed significant reduction in left hind paw swelling following onset of arthritis. Correspondingly, a lesser degree of edema, periosteal new bone formation, periostitis and inflammatory cell infiltration was seen in histological sections of the left hind foot pads of these rats. A similar trend of reduced hind paw swelling was observed in the right hind paws of the same rats and those fed with EOC. Rats fed with GLU alone did not demonstrate these beneficial effects. The present findings demonstrate that a combination of glucosamine and EOC is effective in reducing the histopathological severity of arthritis, probably due to its ability to reduce the inflammatory conditions in CIA.     

Neonatal repetitive needle pricking: Plasticity of the spinal nociceptive circuit and extended postoperative pain in later life

Repetitive exposure of neonates to noxious events is inherent to their health status monitoring in neonatal intensive care units (NICU). Altered basal nociception in the absence of an injury in later life has been demonstrated in ex‐NICU children, but the impact on pain hypersensitivity following an injury in later life is unknown. Also, underlying mechanisms for such long‐term changes are relatively unknown. The objective of this study is to investigate acute and long‐term effects of neonatal repetitive painful skin‐breaking procedures on nociception and to investigate plasticity of the nociceptive circuit. The repetitive needle prick animal model was used in which neonatal rats received four needle pricks into the left hind paw per day during the first postnatal week and control animals received nonpainful tactile stimuli. Repetitive needle pricking during the first week of life induced acute hypersensitivity to mechanical stimuli. At the age of 8 weeks, increased duration of postoperative hypersensitivity to mechanical stimuli after ipsilateral hind paw incision was shown in needle prick animals. Basal nociception from 3 to 8 weeks of age was unaffected by neonatal repetitive needle pricking. Increased calcitonin gene‐related peptide expression was observed in the ipsilateral and contralateral lumbar spinal cord but not in the hind paw of needle prick animals at the age of 8 weeks. Innervation of tactile Aβ‐fibers in the spinal cord was not affected. Ourresults indicate both acute and long‐term effects of repetitive neonatal skin breaking procedures on nociception and long‐term plasticity of spinal but not peripheral innervation of nociceptive afferents. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Activation of extracellular signal-regulated protein kinases 5 in primary afferent neurons contributes to heat and cold hyperalgesia after inflammation

Heat and cold hyperalgesia is a common feature of inflammatory pain. To investigate whether activation of extracellular signal-regulated protein kinase 5 (ERK5), also known as big mitogen-activated protein kinase 1, in primary sensory neurons participates in inflammatory pain, we examined the phosphorylation of ERK5 in the dorsal root ganglion (DRG) after peripheral inflammation. Inflammation induced by complete Freund's adjuvant produced heat and cold hyperalgesia on the ipsilateral hind paw and induced an increase in the phosphorylation of ERK5, mainly in tyrosine kinase A-expressing small- and medium-size neurons. In contrast, there was no change in ERK5 phosphorylation in the spinal dorsal horn. ERK5 antisense, but not mismatch, oligodeoxynucleotide decreased the activation of ERK5 and suppressed inflammation-induced heat and cold hyperalgesia. Furthermore, the inhibition of ERK5 blocked the induction of transient receptor potential channel TRPV1 and TRPA1 expression in DRG neurons after peripheral inflammation. Our results show that ERK5 activated in DRG neurons contribute to the development of inflammatory pain. Thus, blocking ERK5 signaling in sensory neurons that has the potential for preventing pain after inflammation.     

Influence of age on pain sensitivity in response to paw pressure and formalin injection in rats: a role of nitric oxide

The effect of age on pain response to paw pressure and intraplantar formalin injection in rats is elucidated. Pain responses evoked by mechanical pressure on hind paw and intraplantar injection of formaldehyde (5%) into the hind paw were evaluated in groups of adult, young and aged male Sprague Dawley rats, after intraperitoneal (i.p.) or intracerebroventricular (i.c.v.) injection of L-arginine or NG-nitro-L-arginine methyl ester (L-NAME). Nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining was done in the two groups. The results show that pain response was reduced in the aged rats and enhanced pain response to paw pressure in aged rats only. L-arginine (i.c.v.) had no effect on pain response to paw pressure in the two groups but enhanced biphasic pain response to formalin. L-NAME (i.p. and i.c.v.) suppressed pain response to paw pressure in the two groups. L-NAME (i.c.v.) suppressed pain response to formalin during the acute phase and enhanced it during the late phase. NADPH-diaphorase activity was significantly greater in young rats. In conclusion, pain response is blunted in the aged rats. NO might be involved in mechanical nociception in aged rats and in formalin-induced nociception in both groups. NO blockade has an antinociceptive effect on pain response. Central NO has dual role in pain response evoked by formalin.     

Adult spinal opioid receptor μ1 expression after incision is altered by early life repetitive tactile and noxious procedures in rats

Clinical and experimental data suggests that noxious stimulation at critical stages of development results in long‐term changes on nociceptive processing in later life. Here, we use an established, well‐documented rat model of repetitive noxious procedures closely mimicking the clinical situation in the NICU. In order to understand molecular changes underlying the long‐term consequences of repetitive stimulation of the developing nociceptive system the present study aims to analyze the presence of the µ‐opioid‐receptor‐1 (OPRM1). Neonatal rats received either four needle pricks per day in the left hind‐paw from postnatal day 0–7 as a model of procedural pain in infancy. Control pups were handled in the same way but were instead tactile stimulated, or were left undisturbed. At the age of 8 weeks, all animals received an ipsilateral hind‐paw incision as a model for post‐operative pain, and mechanical sensitivity was tested at multiple time‐points. Before, and 1 or 5 days post‐incision, spinal cord tissue was collected for immunostaining of opioid receptor OPRM1. Semi‐quantitative immunocytochemical analysis of superficial laminae in lumbar spinal dorsal horn revealed that: (1) early life repetitive tactile or noxious procedures do not alter baseline levels of OPRM1 staining intensity and (2) early life repetitive tactile or noxious procedures lead to a decrease in OPRM1 staining intensity 5 days after incision in adulthood compared to undisturbed controls. We conclude that early life repetitive tactile or noxious procedures affect the intensity of OPRM1‐immunoreactivity in the lumbar superficial spinal cord dorsal horn after adulthood injury, without affecting baseline intensity. © 2018 The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc. Develop Neurobiol 78: 417–426, 2018     

脊髓细胞外调节激酶1/2在大鼠后足切割痛中的表达和作用

目的：探讨大鼠后足切割后脊髓ERK的表达情况。方法：以大鼠右后足切割作为急性疼痛模型;用免疫组织化学法测试脊髓磷酸化ERK（pERK）表达情况。ERK抑制剂U0126（1μg）在切割前20min或切割后20min鞘内注射。用von Frey纤维测试大鼠机械性痛敏。结果：大鼠后足切割后1min,在切割侧L4-L5脊髓浅层背侧角（板层Ⅰ和板层Ⅱ）ERK被迅速地激活,并在5min达到峰值,随后恢复到基础值。切割前鞘内给予U0126能显著减轻机械性痛敏,然而,切割后鞘内给予U0126对机械性痛敏的作用并不明显。结论：脊髓ERK在大鼠后足切割痛中产生机械性痛敏发挥了重要的作用。     

福尔马林致痛对大鼠脊髓背角神经元神经肽CCK表达的影响

目的：本实验观察胆囊收缩素（CCK）在福尔马林致痛大鼠感觉信息传递中的作用。方法：用免疫组织化学技术,分别观察阴性对照、生理盐水、福尔马林致痛后1h和福尔马林致痛后3h大鼠脊髓背角的感觉神经元神经肽CCK表达的变化。结果：大鼠足底注射福尔马林1h后,脊髓背角Ⅰ、Ⅱ层神经元CCK表达阳性细胞数明显增加（P〈0．01）,且注射侧阳性细胞数明显高于非注射侧。注射福尔马林1h后,注射侧和非注射侧CCK表达阳性细胞的半定量光密度均值分别是0．397±0．014和0．295±0．007,差异有显著性（P〈0．01）;注射福尔马林3h后,注射侧和非注射侧脊髓背角CCK表达阳性细胞的半定量光密度均值分别是0．366±0．009和0．303±0．005,差异仍有显著性（P〈0．01）。结论：福尔马林致痛大鼠脊髓CCK表达阳性细胞的半定量光密度均值增加,进一步证实CCK在炎性痛信息传递通路的多个环节中参与了痛觉调制。     

橄榄叶提取物对大鼠急性炎症和痛觉过敏的研究

本文采用鹿角菜胶诱导的大鼠急性炎症模型观察橄榄叶提取物(OLE)的抗炎与镇痛作用及对致炎因子TNF-α和IL-1β的mRNA表达。用鹿角菜胶注射到大鼠右后足内,分别于鹿角菜胶注射前30 min和注射后120 min灌胃给予OLE 100、250和500 mg/kg体重。鹿角菜胶处理后测量足跖肿胀度及痛域。用RT-PCR检测足跖垫内组织TNF-α、IL-1β和IL-10的mRNA表达。结果显示,经灌胃给予OLE100、250和500 mg/kg体重,均能明显降低大鼠后足肿胀度,增加痛域,并明显降低致炎因子TNF-α和IL-1β的mRNA表达。本研究提示OLE具有抗炎和镇痛作用,其作用机制可能与抑制TNF-α和IL-1β的mRNA表达有关。     

Levels of collagenolytic activity, β-glucuronidase,and collagen prolyl hydroxylase in paws from rats with developing adjuvant arthritis

The collagenolytic activity associated with insoluble collagen fibers separated from homogenates of inflamed paws from rats with adjuvant arthritis was quantitated using EDTA-sensitive solubilization of hydroxyproline as a measure of activity. Approximately 60% of the solubilized hydroxyproline was associated with dialyzable products. The level of collagenolytic activity in the paws increased with time after the induction of adjuvant arthritis and paralleled to a large extent the development of inflammation in both the adjuvant injected (right) hind paw and in the non-injected, contralateral paw. By day 26, the level of free collagenolytic activity in the injected paw had increased to a level 30 times normal while that in the contralateral paw had increased to a level 10 times normal. Treatment of the residues from the injected paws with trypsin resulted in the activation of a latent collagenolytic activity which, on day 26, accounted for approximately 50% of the total activity. The elevated level of collagen prolyl hydroxylase in the inflamed paw suggested that the rate of collagen synthesis was also increased. The activity of β-glucuronidase increased in the inflamed paw with time after the induction of adjuvant arthritis while that of cathepsin G was elevated as compared to normal in paws removed, 5 but not 22 days after the induction of adjuvant arthritis. The inflamed paw of the adjuvant rat may represent a useful system in which to study the role of collagenolytic enzymes in the destruction of connective tissue by inflammatory lesions.     

Angiotensin-(1–7) attenuates collagen-induced arthritis via inhibiting oxidative stress in rats

The present study was designed to investigate the anti-rheumatic effects and the mechanism of angiotensin (Ang)-(1–7) in rat models with collagen-induced arthritis (CIA). The CIA model was established using male Wistar rats by intradermal injection of bovine collagen-II in complete Freund's adjuvant at the base of the tail. The levels of angiotensin converting enzyme 2 (ACE2)/Ang-(1–7)/Mas receptor (MasR) were reduced in CIA rats. The attenuation of paw swelling and arthritis scores and improvement of indexes of spleen and thymus were done by Ang-(1–7) injection in CIA rats. The increased levels of inflammatory cytokines, such as interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ in the serum and hind paw were blocked by Ang-(1–7) administration. In addition, enhanced NADPH oxidase (Nox) activity, increased levels of superoxide anions and malondialdehyde (MDA), and weakened superoxide dismutase (SOD) activity, were all reversed by treatment with Ang-(1–7). Nox1 overexpression reversed the suppressing effects of Ang-(1–7) on paw swelling and arthritis scores in CIA rats. The Ang-(1–7)-induced improvement in spleen and thymus indexes in CIA rats was abolished by Nox1 overexpression. Nox1 overexpression reversed the inhibitory effects of Ang-(1–7) by increasing IL-1β, IL-6, TNF-α, and IFN-γ levels in the serum and hind paw of CIA rats. These results demonstrated that Nox1 increased the oxidative stress in arthritis, and Ang-(1–7) improved rheumatism in arthritis via inhibiting oxidative stress.

     

Identification of a Receptor for Neuropeptide VGF and Its Role in Neuropathic Pain

VGF (nonacronymic) is a neuropeptide precursor that plays multiple roles in regulation of energy balance, reproduction, hippocampal synaptic plasticity, and pain. Data from a number of pain models showed significant up-regulation of VGF in sensory neurons. TLQP-21, one of the VGF-derived neuropeptides, has been shown to induce a hyperalgesic response when injected subcutaneously into the hind paw of mice. However, the precise role of VGF-derived neuropeptides in neuropathic pain and the molecular identity of the receptor for VGF-derived peptides are yet to be investigated. Here we identified gC1qR, the globular heads of the C1q receptor, as the receptor for TLQP-21 using chemical cross-linking combined with mass spectrometry analysis. TLQP-21 caused an increase in intracellular Ca²⁺ levels in rat macrophages and microglia. Inoculation of TLQP-21-stimulated macrophages into rat hind paw caused mechanical hypersensitivity. The increase in intracellular Ca²⁺ levels in macrophages was attenuated by either siRNA or neutralizing antibodies against gC1qR. Furthermore, application of the gC1qR-neutralizing antibody to rats with partial sciatic nerve ligation resulted in a delayed onset of nerve injury-associated mechanical hypersensitivity. These results indicate that gC1qR is the receptor for TLQP-21 and plays an important role in chronic pain through activation of macrophages. Because direct association between TLQP-21 and gC1qR is required for activation of macrophages and causes hypersensitivity, disrupting this interaction may be a useful new approach to develop novel analgesics.     

Inflammatory infiltration of the trigeminal ganglion after herpes simplex virus type 1 corneal infection.

Following herpes simplex virus type 1 (HSV-1) infection of the cornea, the virus is transmitted to the trigeminal ganglion, where a brief period of virus replication is followed by establishment of a latent infection in neurons. A possible role of the immune system in regulating virus replication and maintaining latency in the sensory neurons has been suggested. We have investigated the phenotype and cytokine pattern of cells that infiltrate the A/J mouse trigeminal ganglion at various times after HSV-1 corneal infection. HSV antigen expression in the trigeminal ganglion (indicative of the viral lytic cycle) increased until day 3 postinfection (p.i.) and then diminished to undetectable levels by day 7 p.i. The period of declining HSV antigen expression. was associated with a marked increase in Mac-1+ cells. These cells did not appear to coexpress the F4/80+ (macrophage) or the CD8+ (T cell) markers, and none showed polymorphonuclear leukocyte morphology, suggesting a possible early infiltration of natural killer cells. There was also a significant increase in the trigeminal ganglion of cells expressing the gamma delta T-cell receptor, and these cells were found almost exclusively in very close association with neurons. This period was also characterized by a rapid and equivalent increase in cells expressing gamma interferon and interleukin-4. The density of the inflammatory infiltrate in the trigeminal ganglion increased until days 12 to 21 p.i., when it was predominated by CD8+, Mac-1+, and tumor necrosis factor-expressing cells, which surrounded many neurons. By day 92 p.i., the inflammatory infiltrate diminished but was heaviest in mice with active periocular skin disease. Our data are consistent with the notion that gamma interferon produced by natural killer cells and/or gamma delta T cells may play an important role in limiting HSV-1 replication in the trigeminal ganglion during the acute stage of infection. In addition, tumor necrosis factor produced by CD8+ T cells and macrophages may function to maintain the virus in a latent state.     

Antiinflammatory effect of a protein kinase C inhibitor (K-252a) on the development of the dextran-induced paw edema in the rat (preliminary results).

The effect of a metabolite of Nocardiopsis sp. as a protein kinase C inhibitor from microbial origin was investigated on the onset and development of dextran-induced paw edema in the rat. It was published that this compound (K-252a) interferes with histamine release from mast cells, while dextran-induced paw and nose edema are induced by vasoactive agents, like histamine etc., released from the disrupted mast cells. The antiinflammatory effect of the K-252a is effectuated by the inhibition of protein kinase C. Groups of male Wistar rats with 180-200 g b.w. were used; each group consisted of 10-10 rats. The following groups were consisted: rats given orally DMSO (control), rats given 1 mg/kg, or 3 mg/kg b.w. of K-252a dissolved in DMSO and given p.o. one hour before dextran injection. Dextran (BDH Chem. LTD, molW: 200.000, England) was injected intraperitoneally in 10% solution, in a dose of one ml/100 g b.w. Volume of the hind leg was measured by a mercury plethysmometer. Time-sequence of the edema was followed. Increase in volume of hind leg paw was related to its 0-min volume in %. Results were analyzed by the Kruskal-Wallis-test. Edema of the legs and noses appeared in each of the control rats in one hour. The 1 mg/kg dose of K-252a retarded the appearance of the edema by 1 hour, the 3 mg/kg dose, however, prevented its onset for 4 hours.(ABSTRACT TRUNCATED AT 250 WORDS)