Inflammation-induced Fos protein expression in the rat spinal cord is enhanced following dorsolateral or ventrolateral funiculus lesions

Previous studies have shown an enhanced expression of Fos protein-like immunoreactivity in the lumbar spinal cord of rats with complete spinal transection following persistent hindpaw inflammation. To further locate the spinal pathways responsible for these effects, we compared the inflammation-evoked Fos expression in rats with bilateral lesions of the dorsolateral (DLFX) or ventrolateral (VLFX) funiculus, and with rats with a sham operation. The results indicate that the number of Fos-labeled neurons was significantly increased in all laminae of the dorsal horn ipsilateral to the inflamed hindpaw and in contralateral deep dorsal horn in both DLFX and VLFX rats compared to sham-operated rats. Moreover, when comparing DLFX and VLFX rats, in the ipsilateral spinal cord, DLFX resulted in more Fos expression in the deep dorsal horn; in contrast, a larger number of Fos-labeled cells in superficial laminae was observed in VLFX rats. These results suggest that modulatory systems, which descend in both DLF and VLF pathways, mediate the enhanced net descending nociceptive inhibition after persistent inflammation, although the supraspinal sites of origin of each pathway are likely functionally diverse.     

Immuno-localization of kynurenine aminotransferase (KAT) in the rat medulla and spinal cord

In the mammalian brain, kynurenine aminotransferase (KAT) is pivotal to the synthesis of kynurenic acid, a preferential antagonist at the strychnine-insensitive NMDA-glycine site. As NMDA receptors are involved in autonomic function, we have examined the immunohistochemical localization of KAT in the medulla and spinal cord of the rat. KAT immunoreactivity (KAT-li) was found throughout these areas, in both glia and neurons. Unlike the mainly astrocytic localization in forebrain structures, KAT-li was predominantly neuronal, notably in areas important for blood pressure and heart rate regulation: ventral medulla, nucleus ambiguus, nucleus of the solitary tract and intramediolateral cell column of the spinal cord. The presence of KAT in these nuclei supports a neuromodulatory role for kynurenic acid in NMDA-mediated autonomic function.     

Fos expression in the spinal cord is suppressed in rats displaying conditioned hypoalgesia.

Two experiments used c-fos expression as a marker of spinal nociceptive processing to study the neural correlates of hypoalgesic responses to conditioned stimuli (CSs) paired with an aversive event. Immunoreactive (ir) neuronal labeling of Fos, the nuclear protein encoded by the c-fos gene, was examined in the spinal cords of rats killed 2 hrs after injection of dilute formalin into a hind paw. Compared with control rats either not conditioned or conditioned in one environment but tested elsewhere, there were significantly fewer Fos-ir neurons in the spinal cords of rats displaying hypoalgesic responses when tested in the presence of aversive CSs. Naloxone abolished hypoalgesic responses and reinstated spinal Fos expression, indicating that aversive CSs activated opioid-based antinociceptive mechanisms. The results confirm that aversive CSs produce hypoalgesia by inhibiting the transmission of ascending nociceptive information. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Double immunofluorescence staining of Fos and Jun in the hypothalamus of the rat

Epidermal growth factor (EGF) triggers rapid and delayed effects on gluconeogenesis, cytosolic (lactate/pyruvate ratio) and mitochondrial (3-hydroxybutyrate/acetoacetate ratio) redox states (Soler, C. and Soley, M., Biochem. J., 294 (1993) 865-872). This study attempts to determine whether the mechanism by which EGF modulates any of these parameters is dependent on the regulation of Na+/H+ exchange and/or Na+/K(+)-ATPase activities. The Na+/H+ exchange was inhibited by either amiloride or the analogue 5-(N,N-hexamethylene)amiloride (HMA), and the Na+/K(+)-ATPase activity was inhibited by ouabain. The delayed EGF inhibition of gluconeogenesis, increase of the lactate/pyruvate ratio and decrease in the 3-hydroxybutyrate/acetoacetate ratio were unaltered in the presence of amiloride, HMA or ouabain. The rapid EGF stimulation of gluconeogenesis was also observed in the presence of HMA or ouabain. Although Na+/H+ exchange and/or Na+/K(+)-ATPase are regulated by EGF, our results indicate that these activities are not required for the effects of EGF on gluconeogenesis and/or cytosolic and mitochondrial redox state.     

Differential expression of Fos protein after transection of the rat infraorbital nerve in the trigeminal nucleus caudalis

To determine the effects of nerve injury on Fos expression, temporal and spatial distributions of Fos-positive neurons in the trigeminal nucleus caudalis were examined after tissue injury for isolation of the infraorbital nerve as controls and transection of this nerve as well as noxious chemical stimulation by formalin injection in adult rats. Fos immunoreactivity was markedly elevated in laminae I and II of the only ipsilateral nucleus caudalis 2 h after these surgical procedures and noxious chemical stimulation. The distributions of Fos-positive neurons were restricted rostro-caudally following formalin injection and tissue injury compared to transection of the infraorbital nerve. One day after tissue injury and nerve transection, however, Fos-positive neurons were distributed bilaterally in laminae III and IV extending rostro-caudally and medio-laterally in this nucleus, and this persisted over the 2-week study period. The number of Fos-positive neurons in the side ipsilateral to nerve transection was markedly less than that in the contralateral side whereas positive neurons in the tissue injured rats were distributed symmetrically along the rostro-caudal axis. There was no difference in the contralateral sides between nerve transection and tissue injury groups. The rostro-caudal level showing reduction in Fos expression corresponded roughly to the sites of central termination of the injured nerve in this nucleus, suggesting a role for the primary afferents in the reduction of Fos expression in laminae III and IV neurons of the ipsilateral nucleus caudalis.     

Resistance to anoxic injury in the dorsal columns of adult rat spinal cord following demyelination

In order to examine the relationship between myelination and sensitivity to anoxia in adult white matter, we studied action potential conduction in the spinal cord dorsal column of adult rats in which focal demyelinating lesions had been produced using ethidium bromide/X-irradiation. Acutely isolated spinal cords from control rats and following demyelination were maintained in vitro at 36 degrees C and compound action potentials were studied following supramaximal stimulation. The compound action potential was totally abolished within 12 min of the onset of anoxia in normal dorsal columns, but was not abolished until 50 min following the onset of anoxia in demyelinated dorsal columns. Compound action potentials showed significantly greater recovery (to 58.1 +/- 12.2% of control amplitude) in demyelinated dorsal columns compared to controls (30.8 +/- 5.3%) following 120 min of reoxygenation. These results show that focal demyelination is associated with reduced sensitivity to anoxia within white matter of the adult spinal cord.     

Pre- versus postinjury effects of intravenous GABAergic anesthetics on formalin-induced Fos immunoreactivity in the rat spinal cord

We evaluated the suppression of spinal Fos-like immunoreactivity (FLI) by i.v. anesthetics in the rat formalin model. Preformalin injection (1.5% subcutaneously) treatment groups included i.v. saline controls and three i.v. GABAergic anesthetic groups (pentobarbital 20 mg/kg, propofol 10 mg/kg, or alphaxalone 1.5 mg/kg; n = 12 per group). After perfusion 2 h postformalin, spinal cords were dissected, sliced at 30 microm, and processed by immunoperoxidase staining with an antibody against the Fos protein. Quantification and determination of the laminar distribution of Fos-labeled nuclei were performed at the L4-5 spinal level ipsilateral to formalin injection. Drug groups demonstrating FLI suppression were comparatively studied in a 5-min postformalin treatment group. Pentobarbital pretreatment failed to suppress FLI. However, significant reductions (percent decrease) of FLI were observed with propofol (63%) and alphaxalone (30%) compared with saline controls. Pre- versus postformalin comparison studies showed that propofol, but not alphaxalone, suppressed FLI more effectively when given preformalin. Given the observed inconsistencies between this study of Fos expression and our previous behavioral study, it is questionable whether anesthetic modulation of noxious stimulus-induced FLI parallels that of behavioral responses. Implications: In this study, we examined whether i.v. general anesthetics (propofol, alphaxalone, and pentobarbital) prevent injury-induced spinal cord changes. We measured spinal Fos protein after rats received anesthetics before versus after a formalin injection. Fos inhibition patterns were inconsistent with behavioral studies of these anesthetics, suggesting that Fos inhibition does not always correlate with behavioral analgesia.     

Effects of MK801 on Fos expression in the rat brainstem after unilateral labyrinthectomy

Unilateral labyrinthectomy (UL) causes ocular and postural asymmetries, which disappear over time in the processes of equilibrium recovery known as vestibular compensation. It has been reported that N-methyl-D-aspartate (NMDA) receptors are involved in vestibular compensation. In the present study, in order to elucidate the NMDA receptor-mediated neural circuit responsible for the development of vestibular compensation, we used Fos expression as a marker of neural activation and examined the effects of MK801, a specific antagonist of NMDA receptors, on UL-induced Fos expression in the rat brainstem. After UL, Fos-like immunoreactive (-LIR) neurons were observed in the ipsilateral medial vestibular nucleus (ipsi-MVe), the contralateral prepositus hypoglossal nucleus (contra-PrH) and the contralateral inferior olive beta subnucleus (contra-IOb). Fos-LIR neurons gradually disappeared in the processes of vestibular compensation. It is suggested that the activation of the ipsi-MVe, the contra-PrH and the contra-IOb neurons after UL are the initial event of vestibular compensation. Intraperitoneal injection of MK801 in the processes of vestibular compensation caused reappearance of UL-induced behavioral deficits. During the decompensation induced by MK801, Fos-LIR neurons appeared in the contra-MVe, the ipsi-PrH and the bilateral-IOB. It is suggested that the contra-MVe, the ipsi-PrH and the bilateral-IOb neurons are inhibited by glutamatergic synapses driving inhibitory neurons via NMDA receptors in the processes of vestibular compensation and that disinhibition of these nuclei induced by MK801 causes decompensation. However, MK801 caused neither Fos expression nor behavioral decompensation after vestibular compensation is accomplished. All these findings that the NMDA receptor-mediated inhibitory modulation in the central vestibular system plays an important role for the initial processes of the development of vestibular compensation.     

Sprouting of primary afferent fibers after spinal cord transection in the rat

After spinal cord injury, hyper-reflexia can lead to episodic hypertension, muscle spasticity and urinary bladder dyssynergia. This condition may be caused by primary afferent fiber sprouting providing new input to partially denervated spinal interneurons, autonomic neurons and motor neurons. However, conflicting reports concerning afferent neurite sprouting after cord injury do not provide adequate information to associate sprouting with hyper-reflexia. Therefore, we studied the effect of mid-thoracic spinal cord transection on central projections of sensory neurons, quantified by area measurements. The area of myelinated afferent arbors, immunolabeled by cholera toxin B, was greater in laminae I-V in lumbar, but not thoracic cord, by one week after cord transection. Changes in small sensory neurons and their unmyelinated fibers, immunolabeled for calcitonin gene-related peptide, were assessed in the cord and in dorsal root ganglia. The area of calcitonin gene-related peptide-immunoreactive fibers in laminae III-V increased in all cord segments at two weeks after cord transection, but not at one week. Numbers of sensory neurons immunoreactive for calcitonin gene-related peptide were unchanged, suggesting that the increased area of immunoreactivity reflected sprouting rather than peptide up-regulation. Immunoreactive fibers in the lateral horn increased only above the lesion and in lumbar segments at two weeks after cord transection. They were not continuous with dorsal horn fibers, suggesting that they were not primary afferent fibers. Using the fluorescent tracer DiI to label afferent fibers, an increase in area could be seen in Clarke's nucleus caudal to the injury two weeks after transection. In conclusion, site- and time-dependent sprouting of myelinated and unmyelinated primary afferent fibers, and possibly interneurons, occurred after spinal cord transection. Afferent fiber sprouting did not reach autonomic or motor neurons directly, but may cause hyper-reflexia by increasing inputs to interneurons.     

Blood-spinal cord barrier function and morphometry after single doses of x-rays in rat spinal cord

PURPOSE: The effects of irradiation on blood-spinal cord barrier (BSCB) function and ultrastructure were evaluated using a rat spinal cord model. METHODS AND MATERIALS: Rats received a single dose of 25 Gy to the cervical spinal cord (C2-T2). At various times following irradiation and before the onset of paralysis, BSCB function was assessed using horseradish peroxidase (HRP) as a vascular tracer, and barrier-related structural changes in the capillaries were evaluated using morphometric techniques. RESULTS: Focal extravasation of HRP was seen at 93 days after irradiation, and extensive extravasation was apparent by 114 days in white matter, but not in gray matter. At 93 days, pathologic changes apparent by light microscopy were very minor in the white matter of the irradiated segment. By 107 days, myelin beading, Wallerian degeneration, edema, and histiocytes were apparent in white matter, and these features became increasingly prominent over the following weeks. No noteworthy changes were seen in gray matter at these times. Electron microscopic examination showed that, during the first 93 days following irradiation, more than half of the endothelial cells in white matter had disappeared (p < 0.05). In terms of the putative vascular pores, no abnormalities in endothelial junctions (the presumed small pore) were found, but there was an increase in the density of endothelial vesicles (a putative form of the large pore) in irradiated white matter (p < 0.001), but not in gray matter. Pericytes, thought to act as a second line of defence in the blood-brain barrier, increased in size but not in number in the irradiated white matter of the spinal cord. CONCLUSION: We suggest that radiation damage to endothelial cells, which form the BSCB prior to the onset of neurological deficit, may play an important role in the pathogenesis of white matter necrosis.     

Spinal root avulsion-induced upregulation of osteopontin expression in the adult rat spinal cord

Osteopontin (OPN) is a secretory adhesive glycoprotein that is expressed in various tissues and plays a role in inflammation and tissue repair. It has been suggested that OPN plays a role in inflammation and wound healing after spinal cord injury; however, the expression of OPN and its function in the spinal cord under normal conditions and following spinal motoneuron injury have not been well characterized. Here we examined the expression of OPN mRNA before and after spinal root avulsion. OPN mRNA was detected at a low level in the normal spinal cord in a Northern blot analysis, but dramatically increased following avulsion. In situ hybridization and immunohistochemical studies demonstrated that OPN was present only in a subset of spinal motoneurons before avulsion. After avulsion, the number of OPN-expressing motoneurons increased, although the total number of motoneurons was reduced. OPN expression also became apparent in activated microglia/macrophages and astrocytes. These data suggest that the upregulation of OPN after spinal root avulsion is involved in two events, the protection of neurons and the post-traumatic inflammatory response in microglia/macrophages and astrocytes.     

Morphine-3-glucuronide (M3G) potentiates noxious stimulus-evoked Fos protein-like immunoreactivity in the rat spinal cord

The effect of morphine-3-glucuronide (M3G) on noxious stimulus-evoked Fos protein-like immunoreactivity in the rat spinal cord were assessed by ABC method. It was found that a dose-dependent increase of Fos-like immunoreactive neurons could be induced by M3G intrathecal injection followed by formaline injection into hindpaw. With high dosage M3G (1.1 x 10(-7) mole), dense Fos-like labelling was found in the superficial and the deep dorsal horn bilaterally, While with low dosage M3G (5.4 x 10(-8) and 1.1 x 10(-8) mole), most of the positively labelled neurons were only found in laminae I and II of the ipsilateral dorsal horn to the injured paw. The above results revealed that M3G exerts a potentiating effect on the noxious stimulus-evoked Fos protein-like immunoreactivity in the rat spinal cord.     

Neurons in the hypothalamic paraventricular nucleus send collaterals to the spinal cord and to the rostral ventrolateral medulla in the rat

A simple and sensitive HPLC method for determination of metronidazole in human plasma has been developed. A step of freezing the protein precipitate allowed an efficient separation of aqueous and organic phases minimizing the noise level and improved therefore the limit of quantitation (10 ng ml(-1) using 1 ml of plasma sample). The separation of compounds was performed on a RP 18 column with acetonitrile-aqueous 0.01 M phosphate solution (15:85, v/v) as mobile phase. Detection was performed by UV absorbance at 318 nm. Metronidazole was well resolved from the plasma constituents and internal standard. An excellent linearity was observed between peak-height ratios plasma concentrations over a concentration range of 0.01 to 10 microg ml(-1). Within-day and between-day precision (expressed by relative standard deviation) and accuracy (mean error in per cent) did not exceed 4% between 1 and 10 microg ml(-1) and 8.3 and 7.2% respectively for the limit of quantitation. The method is suitable for bioavailability and pharmacokinetic studies in humans.     

Suckling and sucrose ingestion suppress persistent hyperalgesia and spinal Fos expression after forepaw inflammation in infant rats

The native TRH receptor (TRH-R), which is a G protein-coupled receptor that signals via the phosphoinositide transduction pathway, has been assumed to be inactive in the absence of agonist. In contrast, a mutant mouse TRH-R (C335Stop TRH-R) was shown previously to exhibit constitutive (or agonist-independent) signaling activity. In this report, we measured signaling activity of TRH-Rs using a protein kinase C-responsive reporter gene instead of formation of inositol phosphate second messenger molecules. Using this more sensitive system, we show that native mouse TRH-Rs exhibit agonist-independent signaling activity that is directly proportional to the number of receptors expressed in COS-1 cells and is inhibited by negative antagonist benzodiazepine drugs. As expected, the basal signaling activity of native TRH-Rs is lower than C335Stop TRH-Rs. Constitutive activity of native TRH-Rs is not peculiar to COS-1 cells in which receptor density is markedly elevated, because it can also be demonstrated in Madin Darby canine kidney cells stably expressing mouse TRH-Rs and GH4C1 cells endogenously expressing rat TRH-Rs. These findings support the thesis that native TRH-Rs oscillate between active and inactive states. We suggest that demonstration of constitutive activity of native receptors may depend on the sensitivity of the signaling assay employed.     

Changes in expression of NR-1 and c-jun mRNA in rat lumbar spinal cord after neonatal common peroneal nerve crush

We have examined the expression of the NR-1 subunit of the glutamate NMDA receptor and the immediate early gene c-jun in lumbar spinal cord following neonatal common peroneal nerve crush. The expression of these two genes was studied up to 12 days post-injury (crush occurring at neonatal day P2). The levels of both NR-1 and c-jun mRNA were increased in spinal cord ipsilateral to the site of crush, the induction of mRNA was shown to occur in a time-dependent manner, peaking at 5 days post-injury. The level of NR-1 mRNA showed the most substantial change following nerve crush, increasing 5 times from 4 h to 5 days post-crush. An increase in expression of NR-1 was also observed in spinal cord contralateral to the injury, although quantitatively this was a smaller effect. These results indicate that early postnatal injury causes a significant increase in the expression of NR-1 mRNA which is most marked at 5 days after injury. This period coincides with that of maximum cell death and indicates that the selective induction of NR-1 could underlie the mechanism of this cell death.