Congenital hypothyroidism alters formalin-induced pain response in neonatal rats

The present study designed to investigate the development of nociceptive circuits upon formalin-induced pain in congenital hypothyroid pups during the first three postnatal weeks. Following induction of maternal hypothyroidism, the offspring pups were received right intraplantar injection of different formalin concentrations at 7, 15, and 23 days of age. Significant reduction in weight gain was observed in PTU-treated offspring from postnatal days 15 up to 23 (P < 0.001). No difference was observed between normal and hypothyroid PND7 pups in total pain intensity score with 0.3% solution of formalin. However, normal pups showed higher total pain score (P < 0.01) during the first phase of 1% formalin injection. PND15 normal pups showed a biphasic pain response with a concentration of 2% formalin injection. Obvious persistence of higher pain intensity was observed in hypothyroid pups after interphase through the 2nd phase (P2) and recovery phase (P3), (P < 0.001). PND23 hypothyroid rats showed slightly biphasic pattern of pain behavior with persistence of lower pain intensity during P2 (2.5% formalin, P < 0.05), (10% formalin, P < 0.001) without any further decline during P3 (P < 0.01, P < 0.001 respectively). In general, the number of flexes + shakes in hypothyroid pups was higher than normal pups in both the early and late phases of the test. Licking activity was intensively expressed only in normal pups during phase 2 at the age of 23 days. In contrast to acute pain, hypothyroidism results to pain hypersensitivity in two weeks old rats whereas weaned rats were hyposensitive to tonic nociceptive stimulation without showing the subsequent recovery phase.     

Hypothyroidism following developmental iodine deficiency reduces hippocampal neurogranin,CaMK II and calmodulin and elevates calcineurin in lactational rats

Developmental iodine deficiency (ID) leads to inadequate thyroid hormone that impairs learning and memory with an unclear mechanism. Here, we show that hippocampal neurogranin, calcium/calmodulin dependent protein kinase II (CaMKII), calmodulin (CaM) and calcineurin (CaN) are implicated in the brain impairment in lactational rat hippocampus following developmental ID and hypothyroidism. Three developmental rat models were created by administrating dam rats with either iodine-deficient diet or propylthiouracil (PTU, 5 ppm or 15 ppm)-added drinking water from gestational day (GD) 6 till postnatal day (PN) 21. Then, the neurogranin, CaMKII, CaM and CaN in the hippocampus were detected with immunohistochemistry and western blotting on PN14 and PN21. The iodine-deficient and hypothyroid pups showed significantly lower level of neurogranin, CaMKII and CaM and significantly increased CaN in hippocampal CA1 and CA3 regions than the controls on PN14 and PN21 (P < 0.05, respectively). Data indicate that, in lactational rats, hippocampal neurogranin, CaMKII, CaM and CaN are involved in the brain impairment by developmental ID and hypothyroidism.     

Differential effects of developmental hypo- and hyperthyroidism on acetylcholinesterase and butyrylcholinesterase activity in the spinal cord of developing postnatal rat pups

The plasticity and vulnerability of the rat spinal cord (SC) during postnatal development has been less investigated compared to other CNS structures. In this study, we determined the effects of thyroid hormonal (TH) deficiency and excess on postnatal growth and neurochemical development of the rat SC. The growth as well as the specific and total activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes of the SC were determined in hypo- and hyperthyroid rat pups at postnatal (P) days P1, P5, P10 and P21 (weaning), and were compared to age-matched untreated normal controls. AChE is a cholinergic synaptic enzyme while BuChE is a metabolic enzyme mainly found in glial cells and neurovascular cells. The SC is rich in somatic motor, autonomic cholinergic neurons and associated interneurons. Daily subcutaneous injection of pups with thyroxine (T4) and administration of antithyroid goitrogen propylthiouracil (PTU) in the litter's drinking water were used to induce hyper- and hypothyroidism, respectively. Enzyme assays were carried out spectrophotometrically at the above-mentioned ages, using SC homogenates with acetylthiocholine-chloride as the substrate, together with specific cholinesterase inhibitors, which specifically target AChE and BuChE. SC weights were significantly lower at P10 and P21 in hypothyroid pups but unchanged in the hyperthyroid ones. Hypothyroidism significantly reduced both specific and total AChE activity in SC of P10 and P21 rat pups, while having no effects on the BuChE activity, although total BuChE activity was decreased due to reduced total tissue weight. In contrast both specific and total AChE activities were markedly and significantly increased (>100%) in the P10 and P21 hyperthyroid pups. However, BuChE specific activity was unaffected by this treatment. The results indicate that hypothyroid condition significantly reduces, while hyperthyroidism increases, the postnatal development of cholinergic synapses, thereby influencing the functional development of this major sensory and motor structure. However, the neurochemical development of glia and other non-neuronal cells, where BuChE is mainly localized, is comparatively unaffected in these abnormal developmental conditions.     

NADPH-diaphorase and cytosolic urea cycle enzymes in the rat spinal cord

Nitric oxide synthase (NOS), argininosuccinate synthetase (ASS), and argininosuccinate lyase (ASL) compose a cyclic pathway to form nitric oxide (NO). These enzymes, however, are localized differentially in most regions of the brain. To find out whether NOS, ASS, and ASL are colocalized in neurons of the spinal cord, we examined the distribution of these enzymes by using a double-labeling procedure combining fluorescent immunohistochemistry with an assay for reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d). Results indicate that neurons in the dorsal horn, the intermediolateral nucleus, and the central canal region were NADPH-d active (+) and NOS-, ASS-, and ASL-like immunoreactive (-LI). In laminae II and III of the dorsal horn, some NADPH-d (+) neurons were ASL-LI (8–30%) but only a few were ASS-LI (0.5–7%). In the nucleus intermediolateralis, a large portion of NADPH-d (+) neurons were ASL-LI (30–60%), whereas only a small portion of NADPH-d (+) neurons were ASS-LI (10–20%). In the central canal region, some NADPH-d (+) neurons were ASL-LI (15–40%), and a few NADPH-d (+) neurons were ASS-LI (3–16%). Thus, the results suggest that, in the nucleus intermediolateralis and the central canal region, NOS, ASS, and ASL are colocalized and form a cyclic pathway to produce NO, whereas, in the dorsal horn, these enzymes are more characteristically localized in different neurons, which may transport the substrates intercellularly. J. Comp. Neurol. 385:616–626, 1997. © 1997 Wiley-Liss, Inc.     

Effect of suckling on NADPH-diaphorase (Nitric oxide synthase, NOS) reactivity and NOS gene expression in the paraventricular and supraoptic nuclei of lactating rats

This study examined the effect of suckling on nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d, a histochemical marker for nitric oxide synthase, NOS) reactivity and neuronal NOS mRNA expression in the paraventricular (PVN) and supraoptic (SON) nuclei of lactating rats. Freely nursing (non-separated) dams and those separated from pups for 12 h and then reunited for 0, 15, 30, 60, 90, 120 and 180 min were used for the study. Dams separated from pups and sacrificed at time zero (without reunion) showed a significant decrease in NADPH-d staining and NADPH-d positive cells as well as in the NOS mRNA expression in the PVN and SON compared to that observed in non-separated dams. Reunion with pups and restoration of suckling significantly increased NADPH-d reactivity after 15, 30, 60 min, but not after 90, 120 and 180 min compared to non-reunited pups-deprived dams. A pattern of NADPH-d reactivity and neuronal NOS mRNA expression indistinguishable from that observed during free lactation was reinstated shortly (15 min) after the restoration of suckling stimulus, suggesting that the NADPH-d reactivity in lactation depends on the presence of the suckling stimulus. These results show that suckling stimulus may play a modulatory role in the regulation of NOS reactivity in the magnocellular neurones of the hypothalamic PVN and SON during lactation.     

Treadmill exercise ameliorates symptoms of methimazole-induced hypothyroidism through enhancing neurogenesis and suppressing apoptosis in the hippocampus of rat pups

Thyroid hormones play a crucial role in new neuron production and maturation during brain development. Physical exercise is known to promote cell survival and functional recovery after brain injuries. In the present study, we investigated the effects of treadmill exercise on short-term memory, spatial learning ability, neurogenesis, and apoptosis in hypothyroidism rat pups. On the 14th perinatal day, the pregnant rats were divided into two groups: the maternal control group and the maternal methimazole (MMI)-treated group. For the induction of hypothyroidism in rat pups, MMI was added to the drinking water (0.02%, wt/vol), from the 14th prenatal day to the 49th postnatal day. After delivery, the male rat pups born from the maternal control group were assigned into the control group and the control and exercise group. The rat pups born from the maternal MMI-treated group were divided into the hypothyroidism-induction group and the hypothyroidism-induction and treadmill exercise group. The rat pups in the exercise groups were forced to run on a motorized treadmill for 30 min once a day, starting on the 22nd postnatal day for 4 weeks. Induction of hypothyroidism during the fetal and early postnatal period showed suppression of neurogenesis and enhancement of apoptosis in the hippocampus. Short-term memory and spatial learning ability were deteriorated in the hypothyroidism rat pups. Treadmill exercise during the postnatal period increased neurogenesis and inhibited apoptosis, and resulted in the improvement of short-term memory and spatial learning ability in the hypothyroidism rat pups.     

Changes of substance P-like immunoreactivity in the dorsal horn are associated with the ‘phasic’ behavioral response to a formalin stimulus

Substance P (SP) has been proposed as a nociceptive transmitter/modulator in the dorsal horn of the spinal cord. Formalin used as a nociceptive stimulus has been shown to increase, in a biphasic manner, the amount of immunoreactive SP in the dorsal horn. The time couruse of the changes in substance P-like immunoreactivity (SPLI) caused by formalin is similar to both the electrical activity of dorsal horn neurons and licking behaviors. The administration of morphine reduces stereotypic behaviors caused by a formalin injection but actually increases the amount of SPLI in the dorsal horn. Therefore, the extent to which SP in the dorsal horn is involved witth nociception as a result of formalin remains uncertain. To test the involvement of SP with chemogenic nociception, we utilized lidocaine to block afferent activity prior to an injection of formalin and studied the time course of behaviors and SPLI changes in the dorsal horn. Our results showed that formalin produced two distinct phases of nociceptive behaviors as measured by stereotypic licking of the injected paw: an acute ‘phasic’ response followed by a longer-lasting ‘subacute’ or ‘tonic’ response. Lidocaine reduced both phases of stereotypic behaviors, but only reduced the first increase of SPLI in the dorsal horn. These results suggested a direct involvement of SPLI in the dorsal horn with only ‘phasic’ behavioral responses to a formalin stimulus.     

Comparative effects of neonatal hypothyroidism and euthyroidism on TRH and substance P content of lumbar spinal cord in saline and PCPA-treated rats

The effects of neonatal thyroidectomy and thyroid hormone replacement therapy on the content of substance P and TRH in the lumbar segment of the rat spinal cord were studied. The peptide content of discrete spinal cord regions removed by punches of frozen serial slices was measured by RIA. Animals receiving T4 replacement therapy were indistinguishable from normal littermates. In hypothyroid animals without PCPA-treatment, levels of TRH and substance P were significantly increased by 100% in the ventral and the dorsal lumbar spinal cord, respectively. Inhibition of serotonin biosynthesis by PCPA increased by 90% the substance P content in the dorsal horn of euthyroid rats and abolished completely the stimulatory effect of hypothyroidism on the TRH content of the ventral horn. These findings suggest the existence of a physiological relationship between substance P and TRH with the serotoninergic system in the rat spinal cord and that thyroid hormone is implicated in the normal development of the peptide-containing neurons in the rat spinal cord.     

Intrinsic firing properties of developing rat superficial dorsal horn neurons

Neonatal superficial dorsal horn neurons exhibit distinct firing properties in response to nociceptive and tactile inputs, but it is not known whether the intrinsic membrane excitability of these neurons changes during the early postnatal period. We have investigated the evoked firing properties of dorsal horn cells in rat spinal cord slices at different postnatal ages (P3, P10 and P21) and found no significant differences in mean firing frequency, spike frequency adaptation, regularity of action potential discharge or rheobase current levels between age groups. These results demonstrate that the intrinsic excitability of superficial dorsal horn neurons remains stable during early postnatal development and suggest that alterations in the synaptic inputs to these cells explain the changes in response to peripheral stimulation.     

Ionotropic glutamate receptors contribute to maintained neuronal hyperexcitability following spinal cord injury in rats

In this study, we examined whether topical treatment of glutamate receptor antagonists attenuate hyperexcitability of lumbar spinal dorsal horn neurons following low thoracic hemisection spinal cord injury in rats. Four weeks after spinal hemisection, neuronal activity in response to mechanical stimuli applied on the peripheral receptive field was significantly increased in three different phenotypes of lumbar spinal dorsal horn neurons: wide dynamic range (WDR), low threshold (LT) and high threshold (HT). Topical application of MK-801 (NMDA receptor antagonist, 50 µg) significantly attenuated the activity of WDR, but not LT and HT neurons; whereas, NBQX (AMPA receptor antagonist, 0.5 and 1 µg) significantly attenuated neuronal activity in all three phenotypes of neurons (*p < 0.05). However, MCPG (group I/II metabotropic glutamate receptor antagonist, 100 µg) had no effect. The present study, in the context of previous work, suggests that ionotropic glutamate receptor activation play critical roles in the maintenance of neuronal hyperexcitability and neuropathic “below-level” pain behavior following spinal hemisection injury.     

Hemorrhages of dorsal root ganglia and sensory disturbance in congenitally hydrocephalic HTX rats]

Recently we reported the frequent occurrence of hemorrhages in the dorsal root ganglia of congenitally hydrocephalic HTX rats, an animal model of human congenital hydrocephalus. Therefore, we hypothesized that sensory disturbance might be present in these rats. In order to evaluate the sensory abnormality quantitatively, we injected 50 microliters of 5% formalin into the footpad of the left hind paw of hydrocephalic (n = 5) and non-hydrocephalic HTX rats (n = 5) and Wistar rats (n = 5) on postnatal days 17-23, and then recorded the licking response time for 60 min after injection. The mean licking time in the early (0-10 min) and late (10-60 min) phases was significantly longer in hydrocephalic HTX rats than in non-hydrocephalic HTX and Wistar rats (P < 0.05). Histologically, hemorrhages in the dorsal root ganglia were found in all five hydrocephalic animals, of which two had shown thalamic degeneration. These findings suggest that lesions in the dorsal root ganglia and thalamus may correspond to the sensory disturbance observed in congenitally hydrocephalic HTX rats.     

Postnatal development of NADPH-diaphorase expression in the visual cortex of the golden hamster

Nitric oxide is an important neuromodulator in the brain and is involved in the development of visual system. But it is not clear how nitric oxide and nitric oxide synthase (NOS) are involved in the developing visual cortex of rodents. Thus we examined the expression of NOS activity in the postnatal developing visual cortex of the golden hamster by using histochemical technique for NADPH-diaphorase (NADPH-d). A heavily stained NADPH-d band was observed in the neuropil of the visual cortex. This NADPH-d band initially appeared in the cortical plate from the day of birth (P0) to postnatal day 4 (P4). From P7 to P21, this band was confined to area 17 and migrated to the deeper layers III-IV and V-VI before it eventually disappeared at P28. Such developmental trends of the band correlated well with the process of formation and establishment of the geniculo-cortical projection patterns. Thus, the areal specific development of the band suggests that NOS is closely related to the cortical differentiation and synaptic formation of the primary visual cortex. On the other hand, monocular eye enucleation on P1 could not alter the appearance of this NADPH-d positive band, indicating a non-activity dependant role of NOS. In addition, differences in the laminar distributions and developmental sequence between the heavily and lightly stained NADPH-d positive neurons during development suggest that they play different roles in the development.     

Influences of both thyroid and bovine growth hormones on substance P, thyrotropin-releasing hormone, serotonin and 5-hydroxyindoleacetic acid contents in the lumbar spinal cord of developing rats

Neonatal hypothyroidism was induced by injection of 131I on the first living day whilst neonatal hyperthyroidism was induced by daily administration of high doses of thyroxine (T4). Following decapitation, segments of the lumbar spinal cord were microdissected by a punch technique. We measured serotonin and 5-hydroxyindoleacetic acid (5-HIAA) contents by high performance liquid chromatography and both substance P and thyrotropin-releasing hormone (TRH) levels by radioimmunoassay. We demonstrated that: (1) neonatal hyperthyroidism decreased substance P and TRH levels in the dorsal and ventral horns respectively, without modifying serotonin and 5-HIAA contents; (2) neonatal hypothyroidism increased the concentration of substance P in dorsal horn, of TRH in ventral horn (confirming our previous work), of serotonin in ventral horn, and of 5-HIAA in both ventral and dorsal horns; (3) T4-replacement therapy abolishes hypothyroid effects on substance P, TRH, and 5-HIAA, but not on 5-HT accumulation; and (4) bovine growth hormone-replacement therapy has no therapeutic action on the hypothyroid-induced accumulation of substance P, TRH, serotonin and 5-HIAA.     

Spinal interleukin-6 contributes to central sensitisation and persistent pain hypersensitivity in a model of juvenile idiopathic arthritis

Pain is the most debilitating symptom in juvenile idiopathic arthritis. As pain correlates poorly to the extent of joint pathology, therapies that control joint inflammation are often inadequate as analgesics. We test the hypothesis that juvenile joint inflammation leads to sensitisation of nociceptive circuits in the central nervous system, which is maintained by cytokine expression in the spinal cord. Here, transient joint inflammation was induced in postnatal day (P)21 and P40 male Sprague-Dawley rats with a single intra-articular ankle injection of complete Freund’s adjuvant. Hindpaw mechanical pain sensitivity was assessed using von Frey hair and weight bearing tests. Spinal neuron activity was measured using in vivo extracellular recording and immunohistochemistry. Joint and spinal dorsal horn TNFα, IL1β and IL6 protein expression was quantified using western blotting. We observed greater mechanical hyperalgesia following joint inflammation in P21 compared to P40 rats, despite comparable duration of swelling and joint inflammatory cytokine levels. This is mirrored by spinal neuron hypersensitivity, which also outlasted the duration of active joint inflammation. The cytokine profile in the spinal cord differed at the two ages: prolonged upregulation of spinal IL6 was observed in P21, but not P40 rats. Finally, spinal application of anti-IL-6 antibody (30 ng) reduced the mechanical hyperalgesia and neuronal activation. Our results indicate that persistent upregulation of pro-inflammatory cytokines in the spinal dorsal horn is associated with neuronal sensitisation and mechanical hyperalgesia in juvenile rats, beyond the progress of joint pathology. In addition, we provide proof of concept that spinal IL6 is a key target for treating persistent pain in JIA.     

Differential effect of thyroid hormone deficiency on the growth of calretinin-expressing neurons in rat spinal cord and dorsal root ganglia

The development of spinal cord or dorsal root ganglia neurons expressing calretinin (CR) was studied in thyroid hormone-deficient rats. Immunocytochemical and morphometric analyses showed that the hypothyroidism induced a significant decrease in the number and size of immunoreactive neurons in the spinal cord, as well as stunted growth and arborization of the axons and dendrites. These alterations were observed at different embryonic ages and persisted during the whole postnatal life. In adult hypothyroid rats, the mean number of CR-positive neurons per spinal cord section (31.2 +/- 2.3 in laminae I and II and 30.5 +/- 5.5 in laminae III-X) was significantly decreased (P < 0.001 and P = 0.024, respectively) compared with adult normal rats (68.7 +/- 8.9 and 50.0 +/- 11.0, respectively). In the peripheral nervous system, hypothyroidism altered the growth of sensory neurons expressing CR protein mainly during embryonic life. In comparison with normal rats, hypothyroid embryonic animals showed not only reduced cell size but also a significantly decreased percentage of CR-positive neurons (6.6 +/- 0. 9% in normal, 2.1 +/- 0.3% in hypothyroid rats, P < 0.001). In contrast, although the size of neurons was reduced in hypothyroid young and adult rats, there was no reduction in the percentage of CR-positive neurons. These results showed that thyroid hormone deficiency altered differentially the development of neurons expressing CR protein in the central and peripheral nervous systems. This suggests that central and peripheral neurons are heterogeneous in their sensitivity to thyroid hormone.     

Release of substance P into the superficial dorsal horn following nociceptive activation of the hindpaw of the rat.

Substance P (SP) has been widely proposed as being involved in the transmission of nociceptive information in the dorsal horn of the spinal cord. Formalin injected into the hindpaw as a nociceptive stimulus has been shown to increase the amount of immunoreactive SP in the dorsal horn, perhaps by decreasing SP release from primary afferent neurons. Much is known concerning the release of SP from tissue slices or from the entire spinal cord in vivo. However, less is known about the release patterns of SP in the superficial dorsal horn during the activation of peripheral nociceptors. In this study, noxious pinch applied to and formalin injection into the hindpaw were used as nociceptive stimuli while a stereotaxic push-pull cannula was used to perfuse the L5 dorsal horn. Experiments were conducted in unanesthetized decerebrate/spinal rats, and radioimmunoassay was used to determine the SP-like immunoreactivity (SPLI) content of collected perfusates. Results demonstrate that graded intensities of noxious mechanical pinch produced progressively increased release of SPLI into the dorsal horn; SPLI release returned to baseline rates following termination of the stimulus. The injection of 100 microliters of 5% formalin into the hindpaw produced a biphasic inhibition of SPLI release 0-40 min and greater than 60 min after formalin injection. The application of a noxious pinch following formalin injection produced an increase in SPLI release which did not return to baseline rates; this may be indicative of production of a hyperalgesic state caused by formalin injection. The results of this study support the concept that formalin injected into the hindpaw activates segmental antinociceptive systems which block SP release and limit nociceptive transmission.(ABSTRACT TRUNCATED AT 250 WORDS)     

NADPH-diaphorase in the developing rat: Lower brainstem and cervical spinal cord,with special reference to the trigemino-solitary complex

A previous study indicated that in adult rat, a distinctive neuronal group in the dorsomedial division of the subnucleus oralis of the spinal trigeminal nucleus (SpVo) and the rostrolateral part of the nucleus of the solitary tract (Sn) is stained for nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), and suggested that the labeled structures are involved with sensorimotor reflexive functions. This study aimed to characterize the developmental expression of NADPH-d in SpVo and Sn, including other areas of the lower brainstem and cervical spinal cord, by means of the enzyme histochemical staining technique, from the prenatal through the postnatal period. On embryonic day 12 (E12), no neurons in the brain were stained for NADPH-d, whereas blood vessels were stained. Labeling in the vessels was consistently present throughout pre- and postnatal periods but decreased with development. On E15, labeled neurons appeared in the dorsomedial part of SpVo and the rostrolateral part of Sn, but not in the other nuclei. The labeled neurons in both nuclei increased in numbers drastically to E17. Postnatally, they tended to increase gradually in Sn, but to decrease slightly in SpVo. The cell size of labeled neurons reached a plateau at E17 in SpVo, but at postnatal day 4 (P4) in Sn. In other nuclei on E17, labeling appeared in the lateral paragigantocellular reticular, intermediate reticular, medullary reticular, pedunculopontine tegmental, and spinal vestibular nuclei, and laminae V, VI, and X of the cervical spinal cord. On E20 and P0, labeling appeared in the dorsal column, laterodorsal tegmental, raphe obscurus, parvocellular reticular, ventral gigantocellular reticular, and parahypoglossal nuclei, and laminae IX of the cervical spinal cord. On P4, labeling appeared in the parabrachial and median raphe nuclei, medial and caudolateral Sn, the magnocellular zone of subnucleus caudalis of the spinal trigeminal nucleus (SpVc), and laminae III/IV of the cervical spinal cord. On P10, labeling appeared in the paratrigeminal and dorsal raphe nuclei, the superficial zone of SpVc, and laminae I/II of the cervical spinal cord. No newly labeled neurons appeared in any nuclei after P14. The very early appearance of NADPH-d staining in SpVo and Sn, which precedes the appearance of NADPH-d elsewhere in the brainstem, suggests that the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) system has an important role for primitive orofacial sensorimotor reflexive functions. Furthermore, the pattern of developmental expression of NADPH-d in SpVo and Sn suggests that the NO/cGMP system is organized in a distinct manner in different nuclei. © 1996 Wiley-Liss, Inc.     

Effects of multiple intrathecal administration of L-arginine with different doses on formalin-induced nociceptive behavioral responses in rats

Objective

To investigate the effects of nitric oxide (NO) with different doses on modulation of inflammatory pain, and its possible mechanisms.

Methods

NO precursor L-arginine (L-Arg) was intrathecally administered in rats at a dose of 10 μg per day (low dose group) or 250 μg per day (high dose group) for a succession of 4 d. Normal saline was applied as a control. Then the rats were subcutaneously injected with formalin (100 μL, 2%) into the right hindpaw, and the nociceptive behavioral responses within 1 h were observed. At 4 h after formalin injection, neuronal NO synthase (nNOS) and c-Fos expression in spinal dorsal horn was examined with immunocytochemistry method.

Results

The subcutaneous injection of formalin evoked biphasic behaviors of licking or biting the injected paw. There was no difference in acute phase of formalin test among the 3 groups, while in tonic phase, the licking and biting time, and the protein levels of nNOS and c-Fos in spinal dorsal horn were significantly decreased in low dose group and increased in high dose group, compared with those in control group.

Conclusion

These results suggest that multiple administration of NO with different doses may produce different effects. On one hand, the low dose of NO can induce antinociception. On the other hand, the high dose of NO can induce pronociception.     

Age-related changes in the spinal cord microglial and astrocytic response profile to nerve injury

Neuropathic pain, arising from nerve injury or secondary to other diseases, occurs in young children as well as adults but little is known about its postnatal development. Neonatal rat pups do not display mechanical allodynia following nerve injury and young rats recover faster from spinal nerve damage. Since both spinal microglia and astrocytes are strongly implicated in the maintenance of persistent pain, we hypothesized that the magnitude and time course of spinal cord glial activation following nerve injury change throughout postnatal development. To test this, we have compared the time course and intensity of the microglial and astrocytic response in the spinal cord dorsal horn at various times following spared nerve injury in postnatal day 3, 10, 21 and adult rats. The levels of the microglial markers OX-42 and IBA-1 and of the astrocytic marker GFAP were analysed using immunohistochemistry and Western blots. We show that in the adult SNI evokes clear dorsal horn microglial activation at 5 days and astrocytic activation at 7 days post surgery. In contrast, SNI in young animals evokes a weak microglial response but a robust astrocytic response with an early onset at day 1 that is not observed in adults, followed by a second activation at day 7. These results highlight the differential development of the glial response to nerve injury which may explain the lack of neuropathic allodynia in young animals.     

Hypothyroidism alters antioxidant defence system in rat brainstem during postnatal development and adulthood

The present investigation was carried out to evaluate alterations in oxidative stress parameter [lipid peroxidation (LPx)] and antioxidant enzyme activities [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)] in rat brainstem in response to neonatal hypothyroidism during development (from birth to 7, 15 and 30 days old) and adulthood (90 days old). Hypothyroidism in rats was induced by feeding the lactating mothers (from the day of parturition till weaning, 25 days old) or directly to the pups with 0.05 % [6-n-propyl 2-thiouracil (PTU)] in drinking water. Increased level of LPx was observed in brainstem of 7 days old hypothyroid rats, accompanied by augmented activities of SOD and GPx. In 15 and 30 days old hypothyroid rat brainstem, a significant decline in LPx was observed. Significantly increased activities of CAT and GPx were observed in 15 and 30 days PTU-treated rats. Decreased level of LPx was observed in brainstem of rats treated with PTU from birth to 30 days followed by withdrawal up to 90 days of age (transient hypothyroidism) as compared to control and persistent treatment of PTU up to 90 days of age. Activities of CAT and GPx were decreased in persistent hypothyroid rats of 90 days old with respect to control and transient hypothyroid rats. On the other hand, SOD activity was decreased in both persistent and transient hypothyroid rats with respect to control rats. These results suggest that the PTU-induced neonatal hypothyroidism modulates the antioxidant defence system during postnatal development and adulthood in brainstem of rats.