(-)-Epigallocatechin gallate attenuates NADPH-d/nNOS expression in motor neurons of rats following peripheral nerve injury.

BACKGROUND: Oxidative stress and large amounts of nitric oxide (NO) have been implicated in the pathophysiology of neuronal injury and neurodegenerative disease. Recent studies have shown that (-)-epigallocatechin gallate (EGCG), one of the green tea polyphenols, has potent antioxidant effects against free radical-mediated lipid peroxidation in ischemia-induced neuronal damage. The purpose of this study was to examine whether EGCG would attenuate neuronal expression of NADPH-d/nNOS in the motor neurons of the lower brainstem following peripheral nerve crush. Thus, young adult rats were treated with EGCG (10, 25, or 50 mg/kg, i.p.) 30 min prior to crushing their hypoglossal and vagus nerves for 30 seconds (left side, at the cervical level). The treatment (pre-crush doses of EGCG) was continued from day 1 to day 6, and the animals were sacrificed on days 3, 7, 14 and 28. Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry were used to assess neuronal NADPH-d/nNOS expression in the hypoglossal nucleus and dorsal motor nucleus of the vagus. RESULTS: In rats treated with high dosages of EGCG (25 or 50 mg/kg), NADPH-d/nNOS reactivity and cell death of the motor neurons were significantly decreased. CONCLUSIONS: The present evidence indicated that EGCG can reduce NADPH-d/nNOS reactivity and thus may enhance motor neuron survival time following peripheral nerve injury.

Mild hypoxic preconditioning attenuates injury-induced NADPH-d/nNOS expression in brainstem motor neurons of adult rats.

Excessive production of nitric oxide (NO) might have detrimental effects on the hypoxia-related neuropathology. This study aimed to test if mild hypoxic preconditioning (MHPC) would attenuate the pathological changes in the brainstem motoneurons having a different functional component after peripheral nerve crush injury (PNCI). Prior to PNCI treatment, young adult rats were caged in the mild hypoxic altitude chamber with 79Torr of the partial oxygen concentration ( pO(2)) (i.e., 0.5atm at 5500m in height) for 4 weeks to adapt the environmental changes. After that, all the animals having successfully crushed both the hypoglossal and vagus nerves (left-side) were allowed to survive for 3, 7, 14, 30 and 60 successive days in normoxic condition. Nicotinamine adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry revealed that MHPC reduces NADPH-d/nNOS expression in the hypoglossal nucleus (HN) and the dorsal motor nucleus of the vagus (DMN) at different time points after PNCI. The morphological findings were further ascertained by Western blot analysis of nNOS and nitrite assay for NO production. Both the morphological and quantitative results peaked at 7 days in HN, whereas for those in DMN were progressively increased up to 60 days following PNCI. The staining intensity of NADPH-d/nNOS(+) neurons, expression of nNOS protein, NO production levels as well as the neuronal loss in HN and DMN of MHPC rats following PNCI were attenuated, especially for those having a longer survival period over 14 days. The MHPC treatment might induce minute amounts of NO to alter the state of milieu of the experimental animals to protect against the PNCI.

Reactive changes of retinal astrocytes and Müller glial cells in kainate-induced neuroexcitotoxicity.

The aim of this study was to investigate reactive changes of astrocytes and Müller glial cells in rats subjected to kainate treatment, which leads to neuronal degeneration in the ganglion cell layer and the inner border of the inner nuclear layer as confirmed by labelling with Fluoro-Jade B, a marker for degenerating neurons and fibres. Both the astrocytes and the Müller glial cells reacted vigorously to kainate injection as shown by their up-regulated expression of nestin, glial fibrillary acidic protein and glutamine synthetase. A major finding was the induced expression of nestin together with glial fibrillary acidic protein beginning at 1 day post-injection of kainate. The marked nestin expression appeared to be most intense at 1 day and was sustained till 2 weeks as compared with the untreated/normal retina. Western blotting analysis confirmed a marked increase in expression of nestin, glial fibrillary acidic protein and glutamine synthetase as compared with untreated/normal retina. Double labelling study revealed that astrocytes and Müller glial cells expressed the radial glia marker nestin, and incorporated bromodeoxyuridine to re-enter into their cell cycle. The induced expression of these proteins in astrocytes and Müller glial cells indicated an induction of gliotic responses and de-differentiation that may be associated with regenerative efforts after kainate-induced injury. Indeed, with the acquisition of an immature molecular profile as manifested by the induced expression of brain lipid-binding protein and doublecortin in astrocytes and Müller glial cells, the potential of these cells to de-differentiate in retinal neurodegeneration is greatly amplified.

Total sleep deprivation inhibits the neuronal nitric oxide synthase and cytochrome oxidase reactivities in the nodose ganglion of adult rats.

Sleep disorders are a form of stress associated with increased sympathetic activity, and they are a risk factor for the occurrence of cardiovascular disease. Given that nitric oxide (NO) may play an inhibitory role in the regulation of sympathetic tone, this study set out to determine the NO synthase (NOS) reactivity in the primary cardiovascular afferent neurons (i.e. nodose neurons) following total sleep deprivation (TSD). TSD was performed by the disc-on-water method. Following 5 days of TSD, all experimental animals were investigated for quantitative nicotinamine adenine dinucleotide phosphate-diaphorase (NADPH-d, a co-factor of NOS) histochemistry, neuronal NOS immunohistochemistry and neuronal NOS activity assay. In order to evaluate the endogenous metabolic activity of nodose neurons, cytochrome oxidase (COX) reactivity was further tested. All the above-mentioned reactivities were objectively assessed by computerized image analysis. The clinical significance of the reported changes was demonstrated by alterations of mean arterial blood pressure (MAP). The results indicated that in normal untreated rats, numerous NADPH-d/NOS- and COX-reactive neurons were found in the nodose ganglion (NG). Following TSD, however, both the labelling and staining intensity of NADPH-d/NOS as well as COX reactivity were drastically reduced in the NG compared with normal untreated ganglions. MAP was significantly higher in TSD rats (136+/-4 mmHg) than in normal untreated rats (123+/-2 mmHg). NO may serve as an important sympathoinhibition messenger released by the NG neurons, and decrease of NOS immunoexpression following TSD may account for the decrease in NOS content. In association with the reduction of NOS activity, a defect in NOS expression in the primary cardiovascular afferent neurons would enhance clinical hypertension, which might serve as a potential risk factor in the development of TSD-relevant cardiovascular disturbances.

Microglia/macrophages responses to kainate-induced injury in the rat retina.

The present study was aimed to elucidate how retinal microglia/macrophages would respond to neuronal death after intravitreal kainate injection. An increased expression of the complement receptor type 3 (CR3) and an induction of the major histocompatibility complex (MHC) class II and ED-1 antigens were mainly observed in the inner retina after kainate injection. Prominent cell death revealed by Fluoro Jade B (FJB) staining and ultrastructural examination appeared at the inner border of the inner nuclear layer (INL) at 1 day post-injection. Interestingly, some immunoreactive cells appeared at the outer segment of photoreceptor layer (OSPRL) at different time intervals. Our quantitative analysis further showed that CR3 immunoreactivity was drastically increased peaking at 7 days but subsided thereafter. MHC class II and ED-1 immunoreactivities showed a moderate but steady increase peaking at 3 days and declined thereafter. Double labeling study further revealed that retinal microglia/macrophages expressed concurrently CR3 and ED-1 antigens (OX-42+/ED-1+) or MHC class II molecules (OX-42+/OX-6+) and remained branched in shape at early stage of kainate challenge. By electron microscopy, microglia/macrophages with CR3 immunoreactivity displayed abundant cytoplasm containing a few vesicles and phagosomes. Other cells ultrastructurally similar to Müller cells or astrocytes could also engulf exogenous substances. In conclusion, retinal microglia/macrophages responded vigorously to kainate-induced neuronal cell death that may also trigger the recruitment of macrophages from neighboring tissues and induce the phagocytotic activity of cells other than retinal microglia/macrophages.

Ultrastructural identification of a sympathetic component in the hypoglossal nerve of hamsters using experimental degeneration and horseradish peroxidase methods.

We employed experimental degeneration, tract-tracing with wheatgerm agglutinin conjugated with horseradish peroxidase (WGA-HRP) and electron microscopy to explore the postganglionic sympathetic fibers in the hypoglossal nerve of hamsters. Quantitative results of normal untreated animals at the electron microscopic level showed the existence of unmyelinated fibers, which made up about 20% of the total fibers in the nerve, being more numerous on the left side. The nerve fibers were preferentially distributed at the periphery of the nerve. Following superior cervical ganglionectomy, most of the unmyelinated fibers underwent degenerative changes. Tract-tracing studies showed that some of the unmyelinated fibers were labeled by WGA-HRP injected into the superior cervical ganglion (SCG). It is suggested that the unmyelinated fibers represent the postganglionic sympathetic fibers originated from the SCG.

Melatonin restores the cytochrome oxidase reactivity in the nodose ganglia of acute hypoxic rats.

This study aimed to elucidate whether melatonin would exert beneficial effects on the neuronal functions of the nodose ganglion (NG) following acute hypoxic insult. The cytochrome oxidase (COX) and the nicotinamine adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry along with the nitric oxide synthase (NOS) immunofluorescence were used to examine the metabolic stage and nitric oxide production in nodose neurons respectively. Adult rats were injected intraperitoneally with melatonin at 5 or 100 mg/kg. Hypoxia was achieved by placing the rats into an altitude chamber (PO2 = 43 torr) for 4 hr. The results show that in normal untreated rats, nearly all and about 43% of the NG neurons displayed COX and NOS/NADPH-d reactivities with various staining intensities respectively. However, COX reactivity was drastically decreased while NOS/NADPH-d reactivity was significantly upregulated following hypoxia treatment. In melatonin pretreated rats, the hypoxia-induced reduction of COX reactivity was obviously prevented and the augmentation of NOS/NADPH-d reactivity was successfully suppressed. The deficit in the metabolic stage and the over-activation of NOS would contribute to the generation of oxidative stress. By effectively preventing the metabolic disruption, melatonin may have potential utility in therapeutic treatment of neuronal dysfunctions where oxidative stress is a participant.

Neuronal NADPH-d/NOS expression in the nodose ganglion of severe hypoxic rats with or without mild hypoxic preconditioning.

This study aimed to test the hypothesis that mild hypoxic preconditioning (MHPC)-induced NOS expression would attenuate the neuropathological changes in the nodose ganglion (NG) of severe hypoxic exposure (SHE) rats. Thus, the young adult rats were caged in the altitude chamber for 4 weeks prior to SHE for 4 h to gain hypoxic preconditioning. The altitude chamber was used to set the height at the level from 5500 m (0.50 atm; pO2=79 Torr) to 10,000 m (0.27 atm; pO2=43 Torr) for MHPC and SHE, respectively. The experimental animals were allowed to survive for 0, 7, 14, 30 and 60 successive days, respectively. Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry were used to detect NADPH-d/nNOS reactivity in the NG at various time points following hypoxic exposure. The present results showed that about 38% of the neurons in the NG displayed NADPH-d/nNOS positive [NADPH-d/nNOS(+)] in normoxic rats. In SHE rats, a peak in the percentage (71%) and staining intensity (230%) of NADPH-d/nNOS(+) nodose neurons at 0 day, which then gradually decreased at 7-60 days. About 25% of the nodose neurons died 60 days after SHE. However, in MHPC rats subjected to SHE, NADPH-d/nNOS(+) neurons peaked in the percentage (51%) and staining intensity (171%) at 0 day, which then decreased at 7-60 days. In addition, neuronal survival was markedly increased by MHPC. These results suggested that MHPC might have a neuroprotective effect that reduces the susceptibility of the nodose neurons to NOS mediated neuropathy subsequent to SHE.

Reactive changes of interstitial glia and pinealocytes in the rat pineal gland challenged with cell wall components from gram-positive and -negative bacteria.

Lipopolysaccharide (LPS), the major proinflammatory component of gram-negative bacteria, is well known to induce sepsis and microglial activation in the CNS. On the contrary, the effect of products from gram-positive bacteria especially in areas devoid of blood-brain barrier remains to be explored. In the present study, a panel of antibodies, namely, OX-6, OX-42 and ED-1 was used to study the response of microglia/macrophages in the pineal gland of rats given an intravenous LPS or lipoteichoic acid (LTA). These antibodies recognize MHC class II antigens, complement type 3 receptors and unknown lysosomal proteins in macrophages, respectively. In rats given LPS (50 microg/kg) injection and killed 48 h later, the cell density and immunoexpression of OX-6, OX-42 and ED-1 in pineal microglia/macrophages were markedly increased. In rats receiving a high dose (20 mg/kg) of LTA, OX-42 and OX-6, immunoreactivities in pineal microglia/macrophages were also enhanced, but that of ED-1 was not. In addition, both bacterial toxins induced an increase in astrocytic profiles labelled by glial fibrillary acid protein. An interesting feature following LPS or LTA treatment was the lowering effect on serum melatonin, enhanced serotonin immunolabelling and cellular vacuolation as studied by electron microscopy in pinealocytes. The LPS- or LTA-induced vacuoles appeared to originate from the granular endoplasmic reticulum as well as the Golgi saccules. The present results suggest that LPS and LTA could induce immune responses of microglia/macrophages and astroglial activation in the pineal gland. Furthermore, the metabolic and secretory activity of pinealocytes was modified by products from both gram-positive and -negative bacteria.

Differential expression of calcitonin gene-related peptide (CGRP) and choline acetyltransferase (ChAT) in the axotomized motoneurons of normoxic and hypoxic rats.

We employed a double injury model (axotomy along with hypoxia) to determine how nerve injury and hypoxic insult would affect the expression of calcitonin gene-related peptide (CGRP) and choline acetyltransferase (ChAT) in the hypoglossal nucleus (HN) and nucleus ambiguus (NA). Adult rats were subjected to unilateral vagus and hypoglossal nerve transection, following which half of the animals were kept in an altitude chamber (PO2=380 Torr). The immunoexpression of CGRP and ChAT (CGRP-IR/ChAT-IR) were examined by quantitative immunohistochemistry at 3, 7, 14, 30 and 60 days post-axotomy. The results revealed that CGRP-IR in the HN was increased at 3 days but decreased to basal levels at 7 days following nerve injury. The decline was followed by a second rise in CGRP-IR at 30 days post-axotomy, followed again by a return to basal levels at 60 days. In the NA, CGRP-IR was up-regulated at 3 days and remained increased for up to 60 days after nerve injury. Animals treated with a double injury showed a greater CGRP-IR than normoxic group in both nuclei at all post-axtomized periods. In contrast to CGRP, ChAT-IR was markedly reduced in the HN and NA at 3 days reaching its nadir at 14 days following nerve injury. Hypoxic animals showed a stronger reduction of ChAT-IR in both nuclei at all post-axtomized periods. Results of cell counting showed that neuronal loss was somewhat obvious in hypoxic HN than that of normoxic ones. The present results suggest that up-regulation of CGRP-IR may exert its trophic effects while down-regulation of ChAT-IR may correlate with the poor neurotransmission within the injured neurons. It is speculated that the enhanced expression of CGRP-IR and the pronounced reduction of ChAT-IR in hypoxic rats may result from a drastic shift of intracellular metabolic pathways, which in turn could lead to more metabolic loading to the severely damaged neurons following the double insult.

A time course analysis of the changes in neuropeptide Y immunoreactivity in the rat cuneate nucleus following median nerve transection.

Using median nerve injury and immunocytochemical methods, we examined the temporal changes in neuropeptide Y (NPY) expression in the cuneate nucleus (CN) in rats following median nerve transection. Under normal circumstances, neuropeptide Y-immunoreactive (NPY-IR) fibers was not detectable in the CN. A few NPY-IR fibers were observed in the ipsilateral CN 5 days after the median nerve transection, and peaked at 4 weeks. Thereafter, they were gradually returned to nearly control level after 16 weeks. Quantitative evaluation showed that the mean percentage of area occupied by NPY-IR fibers in entire and three subregions of the CN at 4 weeks were significantly higher than that at other post-operated time points, respectively. The present ultrastructural observations in the middle region of CN showed that the significantly increased NPY immunoreactivity was confined only in the myelinated axons and terminals but not detected in the dendrites, somata, and glial cells. The NPY-IR terminals made axodendritic synaptic contacts with unlabeled elements. The present results indicate that the time course of the increase of NPY immunoreactivity is similar to c-Fos expression as described in a previous study. It is speculated that the increased NPY in the CN after axotomy may affect the excitability of postsynaptic cuneate neurons, however, the functional interaction between NPY and c-Fos-IR neurons needs to be further studied.

Green tea polyphenol (-)-epigallocatechin gallate attenuates the neuronal NADPH-d/nNOS expression in the nodose ganglion of acute hypoxic rats.

Recent studies have shown that (-)-epigallocatechin gallate (EGCG), one of the green tea polyphenols, has a potent antioxidant property. Nitric oxide (NO) plays an important role in the neuropathogenesis induced by brain ischemia/reperfusion and hypoxia. This study aimed to explore the potential neuroprotective effect of EGCG on the ganglionic neurons of the nodose ganglion (NG) in acute hypoxic rats. Thus, the young adult rats were pretreated with EGCG (10, 25, or 50 mg/kg, i.p.) 30 min before they were exposed to the altitude chamber at 10,000 m with the partial pressure of oxygen set at the level of 0.27 atm (pO2=43 Torr) for 4 h. All the animals examined were allowed to survive for 3, 7, and 14 successive days, respectively, except for those animals sacrificed immediately following hypoxic exposure. Nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry were carried out to detect the neuronal NADPH-d/nNOS expression in the NG. The present results show a significant increase in the expression of NADPH-d/nNOS reactivity in neurons of the NG at various time intervals following hypoxia. However, the hypoxia-induced increase in NADPH-d/nNOS expression was significantly depressed only in the hypoxic rats treated with high dosages of EGCG (25 or 50 mg/kg). These data suggest that EGCG may attenuate the oxidative stress following acute hypoxia.

Nitrous oxide suppresses tonic and phasic nociceptive behaviors but not formalin-induced c-Fos expression in the rat spinal cord dorsal horn.

BACKGROUND: The aim of this study was to investigate the anesthetic and analgesic effects of subanesthetic concentration of nitrous oxide and to compare these effects with halothane and fentanyl. METHODS: The antinociceptive effects were assessed in male Sprague-Dawley rats by behavioral responses to phasic and tonic nociceptive stimulations and biochemical index of pain, formalin-induced Fos-like immunoreactivity (Fos-LI), in spinal cord dorsal horn. Neurological functions (proprioception, mental status and motor function) were monitored to determine whether or not behavioral responses were impaired by anesthetic action of the treatment. Four groups of rats treated with: (1) saline, (2) 75% nitrous oxide (0.5 MAC), (3) 0.5% halothane (0.5 MAC) and (4) fentanyl 100 micrograms/kg were subject either to graded intensity of CO2 laser stimulation (phasic pain) or s.c. injection of 50 microliters 2.5% formalin (tonic pain) in two separate studies. All rats in the tonic pain study were killed for immunohistochemistry at 60 min after formalin injection. Maximal counts of Fos-LI labelled neurons in rat spinal cord dorsal horn were compared according to the laminar distribution. RESULTS: We found that all rats exhibited normal righting reflexes regardless of whatever treatment. Nitrous oxide and halothane greatly impaired mental status and motor function, indicating that both agents could induce a modest degree of sedation and paresis at subanesthetic concentrations. Fentanyl increased the threshold level to noxious thermal stimulation, and reduced the formalin-induced licking/biting behaviors and the number of Fos-LI labelled neurons which are predominantly found in the neck of the dorsal horn. Nitrous oxide and halothane increased the thermal nociceptive threshold, suppressed licking/biting behavior in both early and late phases of the formalin test. Unlike fentanyl, nitrous oxide and halothane failed to suppress c-fos expression. The extent and pattern of nitrous oxide-induced antinociception was identical to halothane, which is known to have little or no analgesic effect. The lack of attenuated biochemical response to tonic pain stimulation may suggest that nitrous oxide fails to suppress spinal sensitization despite its potent inhibition on behavioral hyperalgesia. CONCLUSIONS: These findings suggest that, at the spinal level, subanesthetic concentration of nitrous oxide greatly attenuates nociceptive behaviors mainly due to a non-selective suppression of behavioral responses that are commonly associated with inhalation anesthetic drugs.

Subcellular distributions of calcitonin gene-related peptide (CGRP)-like immunoreactivity in the subcommissural organ of the golden hamster (Mesocricetus auratus).

Immuno-electron microscopy specifically enhanced with silver staining has been used to demonstrate the localization of calcitonin gene-related peptide (CGRP) in the ependymocytes of the hamster subcommissural organ (SCO). Hamster SCO consists of the ependymal and hypendymal cell layers, the latter being arranged as rosette-like structure across the posterior commissure (PC) and often arranged with longitudinal axis parallel to the ventricle. All cytoplasmic regions of the ependymal and hypendymal cells were strongly stained with CGRP. In the hypendymal layer, the CGRP positive hypendymal cells were frequently in contact with local blood vessels and arranged in-groups traversing the thick portion of the PC. Ultrastructurally, the CGRP-immunoreaction products were distributed at the dilated cisternae of the rough endoplasmic reticulum (rER) and secretory granules of the ependymal and hypendymal cells. The dilated cisterna of the rER was usually concentrated in the basal region of the ependymal cells and irregular in shape. These dilated cisternae were filled with a flocculent material or finely granular substance, but hardly studded with ribosomes. Labelled secretory granules were abundant in the apical pole of the ependymal cells and discharged their contents into the third ventricle in the form of a thin layer of secretion. This CGRP positive material appeared to constitute the pre-Reissner's fiber (RF). On the basis of the present ultrastructural evidences, we proposed that ependymocytic CGRP in SCO may be synthesized and stored in the cisternae of rER, then released and incorporated into the RF in the third ventricle through the secretory granules. The abundant amount of CGRP in ependymocytes of SCO and RF in the third ventricle suggests a significant endocrine function of CGRP in hamster SCO.

Regional heterogeneity in immunoreactive macrophages/microglia in the rat pineal gland.

Using specific macrophage antibodies (OX-42, OX-6, ED-1 and ED-2), this study examined the distribution of macrophages/microglia in the pineal gland of adult rats. Except for ED-2, all antibodies labeled distinct subpopulations of macrophages/microglia in the gland; ED-2 labeling was hardly detectable. The quantitative study showed that the pineal macrophages/microglia (PMM) expressing complement type 3 receptors (OX-42) were more numerous than those expressing the major histocompatibility complex class II antigen (OX-6) or unknown cytoplasmic/lysosomal antigens (ED-1). The PMM were ubiquitous, especially the OX-42 labeled cells which were distributed from the dorsal to the ventral aspect of the gland. The macrophages/microglia labeled with OX-6 or ED-1 were localized mainly in the intermediate portion of the pineal gland. Immunolabeled cells were sparsely distributed in the distal portion of the pineal gland. A notable feature was that the OX-6 labeled macrophages/microglia showed a proximal-distal gradient in cell density. Another interesting feature was the occurrence of prominent cell aggregations around the larger blood vessels. These cells were mostly round and exhibited different immunoreactivity. Confocal microscopic study with triple immunolabeling further revealed that individual PMM cell possessed two or more different antigens (ED-1+/OX-6+, OX-42+/OX-6+ or OX-42+/ED-1+). Remarkably, a large population co-expressed ED-1+/OX-6+/OX-42+. The present results show that the expression of immunoreactive molecules in PMM varies in topographical distribution of the cells. It is suggested that this may be linked to their immunoregulatory functions in the gland.

Upregulation of NMDA receptor and neuronal NADPH-d/NOS expression in the nodose ganglion of acute hypoxic rats.

Nitric oxide may serve as a neuronal messenger in the regulation of cardiorespiratory function via the N-methyl-D-aspartate (NMDA) receptor-mediated neuronal nitric oxide synthase (nNOS) activation. Since hypoxic stress would drastically influence the cardiorespiratory function, the present study aimed to examine if the expression of nNOS and NMDA receptor subunit 1 (NMDAR1) in the nodose ganglion (NG) would alter under different extents of hypoxia treatment. The nicotinamine adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry, nNOS and NMDAR1 immunofluorescence were used to examine nNOS and NMDAR1 expression in the NG following exposing of adult rats in the altitude chamber (0.27 atm, PO(2)=43 torr) for 2 and 4 h. The present results showed that NADPH-d, nNOS and NMDAR1 reactivities were co-localized in the NG under normoxic and hypoxic environment. Quantitative evaluation revealed that about 43% of neurons in the NG showed positive response for NADPH-d/nNOS and NMDAR1 reactivities. However, in animals subjected to hypoxia, both the percentage and the staining intensity of NADPH-d/nNOS and NMDAR1 labeled neurons were drastically increased. The percentage of NADPH-d/nNOS and NMDAR1-immunoreactive neurons in the NG was raised to 68% as well as 77%, respectively, following 2 and 4 h of hypoxic exposure. The magnitude of up-regulation was positively correlated with the duration of hypoxic periods. No significant cell loss was observed under this experimental paradigm. These findings suggest that different extents of hypoxia might induce the higher expression of nNOS and NMDAR1 in the NG, which could contribute to the neuronal integration as responding to the different physiological demands under hypoxic stress.

Microglial distribution and apoptosis in fetal rat brain.

By histochemical and immunocytochemical techniques, this study aimed to determine the possible involvement of apoptosis in regulating the microglial distribution in fetal rat brain. While microglial cells were labeled with the isolectin Griffonia simplicifolia (GSA I-B4), apoptotic cells were detected by using terminal transferase-mediated dUTP nick end-labeling (TUNEL). TUNEL-labeled cells occurred mainly in the dorsal midline along its rostral-caudal axis of the brain where lectin-labeled microglia were also observed. Occasional TUNEL-labeled cells were observed in the intermediate zone lateral to the striatum (IZS) where lectin-labeled microglia were common from embryonic day 16 (E16) onwards. Some of lectin-labeled microglia showing different morphological forms ingested TUNEL-labeled bodies. In contrast, lectin-labeled microglia showing signs of apoptosis appeared to be lacking. These results clearly demonstrated that lectin-labeled microglia were distributed in areas with and without the occurrence of a large concentration of TUNEL-labeled cells. Our studies suggest that microglia in fetal rat brain will undergo differentiation and activation rather than apoptotic death to govern their population.

Changes in c-Fos protein expression in the rat cuneate nucleus after electric stimulation of the transected median nerve.

In this study we investigate temporal changes in Fos expression in cuneate neurons after a high-threshold electrical stimulation of the transected median nerve in rats. Two hours after injury of the median nerve when given electrical stimulation, c-Fos-immunoreactive (c-Fos-IR) cells were barely detected in the ipsilateral cuneate nucleus (CN). A few c-Fos-IR cells, however, were observed in the ipsilateral CN at 5 days. A marked increase in c-Fos-IR cells was observed at 2, 3, and 4 weeks, but levels subsided thereafter. Labeled cells were totally diminished by 16 weeks. The statistical analysis showed that the mean density of c-Fos-IR cells throughout the CN at 4 weeks was significantly higher than at other post-surgical time points, except for 3 weeks. Furthermore, the mean density of c-Fos-IR cells in the middle region of the CN was markedly higher than in other areas of the nucleus. The mean density of c-Fos-IR cells in the middle region at 4 weeks (mean density = 35.9 +/- 3.0 cells/section) was considerably higher than at other time points. Combined retrograde Fluorogold (FG) labeling and c-Fos immunocytochemistry showed that throughout the CN about 60% (2270/3652) of the c-Fos-IR cells contained FG, confirming that they were cuneothalamic projection neurons (CTNs). Moreover, the percentage of double-labeled cells in the middle region at 2 weeks (78.9 +/- 0.6%) was significantly greater than at 3 (70.2 +/- 3.4%) and 4 weeks (66.0b +/- 1.4%) after injury. Although the mechanism leading to the vigorous c-Fos expression in the CTNs following the electrical stimulation of the transected median nerve remains unclear the hyperexcitable CTNs may transmit the neuropathic nociceptive sensation to the thalamus after the median nerve injury.

Melatonin attenuates the neuronal NADPH-d/NOS expression in the nodose ganglion of acute hypoxic rats.

Excessive production of nitric oxide (NO) may play a detrimental role in the process of hypoxia-related neuropathology. This study explored whether treatment with melatonin would attenuate the neuropathological changes in the vagal ganglia following a severe hypoxic insult. Thirty minutes prior to hypoxia treatment, young adult rats were pre-treated with melatonin at 5. 25 or 100 mg/kg injected intraperitoneally. Hypoxia was achieved by subjecting the rats to a barometric pressure of 0.2 atm (PO2 = 43 Torr) for 4 hr in an altitude chamber. Nicotinamine adenine dinucleotide phosphatediaphorase (NADPH-d) histochemistry combined with the neuronal nitric oxide synthase (nNOS) immunohistochemistry were used to detect the NADPH-d/nNOS reactivity in the nodose ganglion (NG) at various time points following the hypoxic exposure. In normal untreated rats, about 43% of the neurons in the NG displayed NADPH-d/nNOS reactivity. Following hypoxic exposure, both the percentage and the staining intensity of NADPH-d/nNOS positive neurons in the NG were markedly increased, but these were reduced in longer surviving animals. Quantitative analysis of cell counts revealed that about 17% of the neurons died at 14 days after hypoxia treatment. However, in hypoxic rats given different doses of melatonin pretreatment, neuronal death as well as the frequency and staining intensity of NADPH-d/nNOS reactivity of the nodose neurons were significantly decreased. The effect of melatonin on neuronal survival and NADPH-d/ nNOS expression was dose-dependent. It is therefore suggested that melatonin exerts a neuroprotective effect and may serve as a potential therapeutic strategy for prevention and/or reducing the susceptibility of nodose neurons to NO-mediated hypoxic neuropathy.

Neurogenesis of cuneothalamic neurons and NO-containing neurons in the cuneate nucleus of the rat.

The genesis of the cuneothalamic neurons (CTNs) in the rat cuneate nucleus was determined by a double-labeling method using 5'-bromodeoxyuridine (BrdU), the thymidine analogue, and Fluoro-Gold (FG), a retrograde fluorescent tracer. BrdU-positive cells were observed in the cuneate nucleus in all rats receiving BrdU injection at embryonic days (E) E13--E16; none was detected in rats given BrdU injection at E12. At E13 and E14, BrdU-positive cells were randomly distributed. However, at E15, the number of BrdU-positive cells was clearly reduced and the majority of them was located at the dorsolateral or peripheral region of the nucleus. FG/BrdU double-labeling study showed the existence of BrdU-labeled CTNs when the mother rat received BrdU injection at E13 and E14, being more numerous at E13 in which the neurons were scattered throughout the nucleus. At E14, however, the majority of the BrdU-labeled CTNs were located superficially in the nucleus. Double-labeled cells were undetected in rats that had been exposed to BrdU at E15 and E16. Quantitative data showed that the majority (ca 70-80%) of the CTNs were generated at E13, and were markedly decreased at E14 (ca 4-6%). Using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry coupled with BrdU immunohistochemistry, we have shown the NADPH-d/BrdU double-labeled neurons in the nucleus between E13 and E15, with the majority of them occurring at E14, but absent at E16. The present results suggest that the CTNs are generated prior to the NO-containing neurons in the cuneate nucleus.