Enkephalin knockout male mice are resistant to chronic mild stress

Enhanced stress reactivity or sensitivity to chronic stress increases the susceptibility to mood pathologies such as major depression. The opioid peptide enkephalin is an important modulator of the stress response. Previous studies using preproenkephalin knockout (PENK KO) mice showed that these animals exhibit abnormal stress reactivity and show increased anxiety behavior in acute stress situations. However, the consequence of enkephalin deficiency in the reactivity to chronic stress conditions is not known. In this study, we therefore submitted wild‐type (WT) and PENK KO male mice to chronic stress conditions, using the chronic mild stress (CMS) protocol. Subsequently, we studied the CMS effects on the behavioral and hormonal level and also performed gene expression analyses. In WT animals, CMS increased the expression of the enkephalin gene in the paraventricular nucleus (PVN) of the hypothalamus and elevated the corticosterone levels. In addition, WT mice exhibited enhanced anxiety in the zero‐maze test and depression‐related behaviors in the sucrose preference and forced swim tests. Surprisingly, in PENK KO mice, we did not detect anxiety and depression‐related behavioral changes after the CMS procedure, and even measured a decreased hormonal stress response. These results indicate that PENK KO mice are resistant to the CMS effects, suggesting that enkephalin enhances the reactivity to chronic stress.     

Synthetic peptide TPLVTLFK (octarphin) reduces the corticosterone production by rat adrenal cortex through nonopioid β‐endorphin receptor

The synthetic peptide octarphin (TPLVTLFK) corresponding to the sequence 12–19 of β‐endorphin, a selective agonist of nonopioid β‐endorphin receptor, was labeled with tritium to a specific activity of 29 Ci/mmol. [³H]Octarphin was found to bind to high‐affinity naloxone‐insensitive binding sites on membranes isolated from rat adrenal cortex (K_d = 35.7 ± 2.3 nM, B_max = 41.0 ± 3.6 pmol/mg protein). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but to α‐endorphin, γ‐endorphin, [Met⁵]enkephalin, and [Leu⁵]enkephalin as well. At the same time, the [³H]octarphin‐specific binding with adrenal cortex membranes was inhibited by unlabeled β‐endorphin (K_i = 32.9 ± 3.8 nM). Octarphin at concentrations of 10^?9–10^?6 M was found to inhibit the adenylate cyclase activity in adrenocortical membranes, whereas intranasal injection of octarphin at doses of 5 and 20 µg/rat was found to reduce the secretion of corticosterone from the adrenals to the bloodstream. Thus, octarphin decreases the adrenal cortex functional activity through the high affinity binding to nonopioid receptor of β‐endorphin. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Region-dependent G-protein activation by mu-, delta 1- and delta 2-opioid receptor agonists in the brain: comparison between the midbrain and forebrain

The ability of selective mu- ([D-Ala2, NHPhe4, Gly-ol]enkephalin: DAMGO), delta1- ([D-Pen2, Pen5]enkephalin: DPDPE) and delta2- ([D-Ala2]deltorphin II: DELT II) opioid receptor agonists to activate G-proteins in the midbrain and forebrain of mice and rats was examined by monitoring the binding of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS). The levels of [35S]GTPgammaS binding stimulated by DAMGO in the mouse and rat midbrain were significantly greater than those by DPDPE or DELT II. However, relatively lower levels of stimulation of [35S]GTPgammaS binding by all of the agonists than would have been predicted from the receptor densities were observed in either the limbic forebrain or striatum of mice and rats. The effects of DAMGO, DPDPE and DELT II in all three regions were completely reversed by selective mu-, delta1- and delta2-antagonists, respectively. The results indicate that the levels of mu-, delta1- and delta2-opioid receptor agonist-induced G-protein activation in the midbrain are in good agreement with the previously determined distribution densities of each receptor type. Furthermore, the discrepancies observed in the forebrain might reflect differential catalytic efficiencies of receptor-G-protein coupling.     

Ontogenesis of enkephalin and humoral endorphin in the rat brain

Radioimmunoassay and column chromatography techniques were used to study the postnatal development of two different opioid ligands, humoral endorphin and enkephalin in the rat brain. Similar patterns were observed for both male and female animals during the period examined (from birth to the seventh week of life). Humoral endorphin content of the developing rat brain was found to increase in parallel to enkephalin, exhibiting a ‘lag’ period of 2 weeks. The most dramatic increase in opioid levels was detected during the third week of life; this stage was followed by a gradual change up to the adult levels.     

Proteome of synaptosome‐associated proteins in spinal cord dorsal horn after peripheral nerve injury

Peripheral nerve injury may lead to neuroadaptive changes of cellular signals in spinal cord that are thought to contribute to central mechanisms underlying neuropathic pain. Here we used a 2‐DE‐based proteomic technique to determine the global expression changes of synaptosome‐associated proteins in spinal cord dorsal horn after unilateral fifth spinal nerve injury (SNI). The fifth lumbar dorsal horns ipsilateral to SNI or sham surgery were harvested on day 14 post‐surgery, and the total soluble and synaptosomal fractions were isolated. The proteins derived from the synaptosomal fraction were resolved by 2‐DE. We identified 27 proteins that displayed different expression levels after SNI, including proteins involved in transmission and modulation of noxious information, cellular metabolism, membrane receptor trafficking, oxidative stress, apoptosis, and degeneration. Six of the 27 proteins were chosen randomly and further validated in the synaptosomal fraction by Western blot analysis. Unexpectedly, Western blot analysis showed that only one protein in the total soluble fraction exhibited a significant expression change after SNI. The data indicate that peripheral nerve injury changes not only protein expression but also protein subcellular distribution in dorsal horn cells. These changes might participate in the central mechanism that underlies the maintenance of neuropathic pain.     

Binding of synthetic peptide TPLVTLFK to nonopioid beta‐endorphin receptor on rat brain membranes

The synthetic peptide TPLVTLFK corresponding to the sequence 12–19 of β‐endorphin (referred to as octarphin) was found to bind to high‐affinity naloxone‐insensitive binding sites on membranes isolated from the rat brain cortex (K_d = 2.6 ± 0.2 nM ). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but also to α‐endorphin, γ‐endorphin, [Met⁵]enkephalin, and [Leu⁵]enkephalin, as well. The [³H]octarphin specific binding with brain membranes was inhibited by unlabeled β‐endorphin (K_i = 2.4 ± 0.2 nM ) and a selective agonist of nonopioid β‐endorphin receptor decapeptide immunorphin SLTCLVKGFY (K_i = 2.9 ± 0.2 nM ). At the same time, unlabeled octarphin completely (by 100%) inhibited the specific binding of [³H]immunorphin with membranes (K_i = 2.8 ± 0.2 nM ). Thus, octarphin binds with a high affinity and specificity to nonopioid receptor of β‐endorphin on rat brain cortex membranes. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

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     

Effects of neonatal maternal separation on neurochemical and sensory response to colonic distension in a rat model of irritable bowel syndrome

Early life stress has been implicated as a risk factor for irritable bowel syndrome (IBS). We studied the effect of neonatal maternal separation on the visceromotor response and the expression of c-fos, 5-HT, and its receptors/transporters along the brain-gut axis in an animal model of IBS. Male neonatal Sprague-Dawley rats were randomly assigned to a 3-h daily maternal separation (MS) or nonhandling (NH) on postnatal days 2-21. Colorectal balloon distention (CRD) was performed for assessment of abdominal withdrawal reflex as a surrogate marker of visceral pain. Tissues from dorsal raphe nucleus in midbrain, lumbar-sacral cord, and distal colon were harvested for semiquantitative analysis of c-fos and 5-HT. The expression of 5-HT expression, 5-HT3 receptors, and 5-HT transporter were analyzed by RT-PCR. Pain threshold was significantly lower in MS than NH rats. The abdominal withdrawal reflex score in response to CRD in MS rats was significantly higher with distension pressures of 40, 60, and 80 mmHg. In MS rats, the number of c-fos-like immunoreactive nuclei at dorsal horn of lumbar-sacral spinal cord increased significantly after CRD. 5-HT content in the spinal cord of MS rats was significant higher. In the colon, both 5-HT-positive cell number and 5-HT content were comparable between MS and NH groups before CRD. Post-CRD only MS rats had significant increase in 5-HT content. Protein and mRNA expression levels of 5-HT3 receptors and 5-HT transporter were similar in MS and NH rats. Neonatal maternal separation stress predisposes rats to exaggerated neurochemical responses and visceral hyperalgesia in colon mimicking IBS.     

Interaction of synthetic peptide octarphin (TPLVTLFK) with human blood lymphocytes

The synthetic peptide octarphin (TPLVTLFK) corresponding to the sequence 12–19 of β‐endorphin, a selective agonist of non‐opioid β‐endorphin receptor, was labeled with tritium to specific activity of 29 Ci/mmol. The analysis of [³H]octarphin binding to human T and B lymphocytes separated from normal human blood revealed the existence of one type of high‐affinity binding sites (receptors): K_d 3.0 and 3.2 nM, respectively. Besides unlabeled octarphin, unlabeled β‐endorphin possessed the ability to inhibit the specific binding of [³H]octarphin to Т and B lymphocytes (K_i 1.9 and 2.2 nМ, respectively). Tests of the specificity of the receptors revealed that they are not sensitive to naloxone, α‐endorphin, γ‐endorphin, [Met⁵]enkephalin, and [Leu⁵]enkephalin. Thus, both T and B lymphocytes from normal human blood express non‐opioid receptor for β‐endorphin. Binding of the hormone to the receptor provides a fragment 12–19. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

2',3'-Cyclic nucleotide 3'-phosphohydrolase in the developing chick brain and spinal cord

Developmental changes of the 2',3'-cyclic nucleotide 3'-phosphohydrolase activity in the chick brain and spinal cord are reported. The greater part of increase in enzyme activity occurred between 18 days of incubation and 3 days after hatching in the whole brain, and between 18 and 21 days of incubation in the spinal cord. These periods are those of active myelination in the chick brain and spinal cord, respectively. The possibility was emphasized that 2',3'-cyclic nucleotide 3'-phosphohydrolase can be used as a marker for the myelin sheaths in the developing central nervous system. Comparisons were also made among the developmental changes in the forebrain, midbrain, brain stem, cerebellum, and spinal cord.     

Regulation of hypophysial secretion by endogenous opiates: Humoral endorphin stimulates the release of growth hormone

Humural endorphin, a recently discovered endogenous opioid factor stimulates the release of growth hormone and, to some extent of prolactin, similarly to other endogenous (enkephalin, β-endorphin) and exogenous (morphine) opiates. This stimulatory effect is dose-dependent with peak values at 30 minutes following intraventricular injection to newborn rats. However, in contrast to the other opioid ligands, the effect of humoral endorphin is not blocked in a dose-dependent fashion by naloxone, the potent opiate antagonist. Thus, while moderate doses of naloxone partially inhibit the stimulatory effect, higher doses which completely block morphine, enkephalin and β-endorphin, are ineffective in antagonizing humoral endorphin. This peculiar interaction between naloxone and humoral endorphin resembles the effect of the opiate antagonist on spontaneous release of growth hormone and prolactin, suggesting the involvement of humoral endorphin in the physiological regulation of hypophysial secretion.     

100Hz电针刺受时大鼠脑和脊髓k受体结构特性的改变

西方采用放射配体结合实验研究了１００ＨＺ电针耐受发生发展过程中大鼠脑和脊髓Ｋ受体结构特性的变化。大鼠每天给予１００ＨＺ电针１次,连续７ｄ。分别在电针的第１、３、５、７天取不同脑区进行观察。     

Effect of intrathecal injection of pertussis toxin on substance P, norepinephrine and serotonin contents in various neural structures of arthritic rats.

The possible influence of spinal receptors coupled to Gi/Go regulatory proteins on chronic pain adaptive processes of neural tissues was investigated in normal and arthritic rats. Pain-suffering animals showed an enhanced immunoreactivity to substance P (ir-SP) in the lumbar spinal cord, pons-medulla oblongata region and thalamus. Norepinephrine (NE) levels were increased in the spinal cord, while serotonin (5-HT) was elevated in both spinal cord and midbrain. The intrathecal injection of 1 micrograms pertussis toxin 6 days before sacrifice of rats produced in these arthritic animals a pronounced reduction of ir-SP in the pons-medulla, midbrain and thalamus, but not in the spinal cord. The level of 5-HT was diminished in dorsal spinal cord and midbrain, whereas NE appeared unchanged. In contrast, the toxin only reduced ir-SP of normal rats in the midbrain, without altering the levels of NE or 5-HT, in all the areas analysed. These results suggest the involvement of certain spinal receptors coupled to Gi/Go transducer proteins in processes leading to the elevation of ir-SP and 5-HT in various neural structures of arthritic rats.     

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     

The mechanical properties of neonatal rat spinal cord in vitro,and comparisons with adult

A number of studies have investigated the mechanical properties of adult spinal cord under tension, however it is not known whether age has an effect on these properties. This is of interest to those aiming to understand the clinical differences between adults and children with spinal cord injury (e.g. severity and recovery), and those developing experimental or computational models for paediatric spinal cord injury. Entire spinal cords were freshly harvested from neonatal rats (14 days) and tested in vitro under uniaxial tension at a range of strain rates (0.2, 0.02, 0.002/s) to a range of strains (2%, 3.5%, 5%), with relaxation responses being recorded for 15 min. These mechanical properties were compared to previously reported data from similar experiments on adult rat spinal cords, and the peak stress and the stress after 15 min of relaxation were found to be significantly higher for spinal cords from adults than neonates (p<0.001). A non-linear viscoelastic model was developed and was observed to adequately predict the mechanical behaviour of this tissue. The model developed in this study may be of use in computational models of paediatric spinal cord. The significant differences between adult and neonatal spinal cord properties may explain the higher initial severity of spinal cord injury in children and may have implications for the development of experimental animal models for paediatric spinal cord injury, specifically for those aiming to match the injury severity with adult experimental models.     

Effect of Chronic Administration of Morphine, U-50, 488H and [D-Pen, D-Pen]enkephalin on the Concentration of cGMP in Brain Regions and Spinal Cord of the Mouse

Bhargava, H. N. and Y. J. Cao. Effect of chronic administration of morphine, U-50,488H and [ -Pen², -Pen⁵]enkephalin on the concentration of cGMP in brain regions and spinal cord of the mouse. Peptides 18(10) 1629–1634, 1997.—The effects of chronic administration and subsequent withdrawal of μ-, κ- and δ-opioid receptor agonists on the levels of cyclic GMP in several brain regions and spinal cord of mice were determined in an attempt to further study the role of NO cascade in opioid actions. The agonists at μ-, κ- and δ-opioid receptor included morphine, U-50,488H and DPDPE, respectively. Tolerance to morphine was associated with highly significant increases in cGMP levels in corpus striatum (41%), cortex (36%), midbrain (73%) and cerebellum (51%) relative to controls. Abstinence caused increases in cGMP levels in corpus striatum (61%) and pons and medulla (45%). Tolerance to U-50,488H resulted in increases in cGMP levels in midbrain (52%) whereas abstinence from U-50,488H increased the cGMP levels in pons and medulla(76%). Tolerance to DPDPE was associated with increases in cGMP levels in hypothalamus (12%) and pons and medulla (33%) but decreases in cerebellum (66%) and spinal cord (58%). Abstinence from DPDPE produced increases in cGMP levels in pons and medulla (14%) but decreases in cerebellum (67%) and spinal cord (50%). Overall treatment with morphine and U-50,488H produced increases in cGMP levels in brain regions whereas DPDPE produced decreases in brain regions and spinal cord. Previous studies have shown that chronic administration of μ- and κ- opioid receptor agonists induce NO synthase (NOS) in certain brain regions and that the inhibitors of NO synthase attenuate tolerance to μ- and κ- but not to δ-opioid receptors agonists. Since activation of NO increases the production of cGMP, the present results demonstrating alterations of cGMP levels by μ-, κ- and δ-opioid receptor agonists are consistent with the behavioral results with NOS inhibitors on tolerance to μ-, κ- and δ-opioid receptor agonists.     

Role of IL‐15 in spinal cord and sciatic nerve after chronic constriction injury: regulation of macrophage and T‐cell infiltration

The release of inflammatory mediators from immune and glial cells either in the peripheral or CNS may have an important role in the development of physiopathological processes such as neuropathic pain. Microglial, then astrocytic activation in the spinal cord, lead to chronic inflammation, alteration of neuronal physiology and neuropathic pain. Standard experimental models of neuropathic pain include an important peripheral inflammatory component, which involves prominent immune cell activation and infiltration. Among potential immunomodulators, the T‐cell cytokine interleukin‐15 (IL‐15) has a key role in regulating immune cell activation and glial reactivity after CNS injury. Here we show, using the model of chronic constriction of the sciatic nerve (CCI), that IL‐15 is essential for the development of the early inflammatory events in the spinal cord after a peripheral lesion that generates neuropathic pain. IL‐15 expression in the spinal cord was identified in both astroglial and microglial cells and was present during the initial gliotic and inflammatory (NFκB) response to injury. The expression of IL‐15 was also identified as a cue for macrophage and T‐cell activation and infiltration in the sciatic nerve, as shown by intraneural injection of the cytokine and activity blockage approaches. We conclude that the regulation of IL‐15 and hence the initial events following its expression after peripheral nerve injury could have a future therapeutic potential in the reduction of neuroinflammation.     

Proteomic analysis of spinal protein expression in rats exposed to repeated intrathecal morphine injection

Repeated administration of morphine for treating severe chronic pain may lead to neuroadaptive changes in the spinal cord that are thought to underlie molecular mechanisms of the development of morphine tolerance and physical dependence. Here, we employed a 2-D gel-based proteomic technique to detect the global changes of the spinal cord protein expression in rats that had developed morphine tolerance. Morphine tolerance at the spinal cord level was induced by repeated intrathecal injections of morphine (20 microg/10 microL) twice daily for 5 days and evaluated by measurements of paw withdrawal latencies and maximal possible analgesic effect at day 5. After behavioral tests, the lumbar enlargement segments of spinal cord were harvested and proteins resolved by 2-DE. We found that eight proteins were significantly up-regulated or down-regulated in spinal cord after morphine tolerance development, including proteins involved in targeting and trafficking of the glutamate receptors and opioid receptors, proteins involved in oxidative stress, and cytoskeletal proteins, some of which were confirmed by Western blot analysis. Morphine-induced expressional changes of these proteins in the spinal cord might be involved in the central mechanisms that underlie the development of morphine tolerance. It is very likely that these identified proteins may serve as potential molecular targets for prevention of the development of morphine tolerance and physical dependence.     

Adrenalectomy affects pain behavior of rats after formalin injection

Stressful stimuli can activate the hypothalamo-pituitary-adrenal-axis and the endogenous opioid system. In addition, corticosterone and opioid release might cause analgesia. This rat study used adrenalectomy for corticosterone withdrawal and naloxone administration for opioid antagonism in order to study pain behavior and hypophyseal hormone release in the plasma after a formalin test. Twelve days before the formalin testing, male Sprague Dawley rats underwent adrenalectomy or sham-adrenalectomy, and non-operated rats were used as reference. The number of flinches and the duration of licking or biting behavior were measured during the early and late phase. In reference and sham-operated rats, injection of formalin 5% resulted in a marked pain behavior in the early and late phase with significant increases in ACTH and corticosterone plasma levels. In adrenalectomized rats, pain behavior was decreased during both phases. Naloxone, administered before the late phase, did not alter pain behavior in sham or reference rats, whereas in adrenalectomized rats pain reactivity returned to those levels observed in reference rats. Beta-endorphin plasma levels above the detection limit were more frequently found in adrenalectomized rats. Thyrotropin and prolactin levels were not different between studied groups. We speculate that the observed reduced pain behavior in adrenalectomized rats after formalin, is the result of an increased production of pro-opiomelanocortin, the pro-drug of both adrenocorticotrophic hormone and beta-endorphin.     

孤啡肽受体对痛觉调控作用的研究进展

孤啡肽受体是继经典的mu阿片受体、kappa阿片受体和delta阿片受体之后发现的又一类新型阿片受体,不仅在结构上具有同上述阿片受体相类似的特征,而且可介导相同或相似的细胞内生物学反应.孤啡肽受体对痛觉反应具有独特的调控模式.一方面,在背根神经节以及脊髓水平,孤啡肽受体主要介导镇痛效应,并且在脊髓水平还与其他阿片受体有协同效应以增强镇痛效果.另一方面,在脊髓上水平,孤啡肽受体往往产生痛敏而拮抗了其他阿片受体的镇痛效应.此外孤啡肽受体对痛觉的调控在不同物种间也表现一定的差异性.这为进一步阐明内源性阿片系统的痛觉调控作用提供一定的理论依据.