Ischemic preconditioning-induced neuroprotection against transient cerebral ischemic damage via attenuating ubiquitin aggregation

Ubiquitin binds to short-lived proteins, and denatured proteins are produced by various forms of injuries. In the present study, we investigated the effect of ischemic preconditioning (IPC) on free ubiquitin and its mutant form (ubiquitin^+ 1) in the gerbil hippocampus induced by transient cerebral ischemia. The animals were randomly assigned to 4 groups (sham-operated-group, ischemia-operated-group, IPC plus (+)-sham-operated-group, and IPC + ischemia-operated-group). IPC was induced by subjecting gerbils to a 2 min of ischemia followed by 1 day of recovery. A significant loss of neurons was observed in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) in the ischemia-operated-groups 5 days after ischemia–reperfusion (I–R). In all the IPC + ischemia-operated-groups, neurons in the SP were well protected. We found that strong ubiquitin immunoreactivity was detected in the SP in the sham-operated-group and the immunoreactivity was decreased with time after I–R. In all the IPC + ischemia-operated-groups, ubiquitin immunoreactivity in the SP was similar to that in the sham-operated group. Moderate ubiquitin^+ 1 immunoreactivity was detected in the SP of the sham-operated-group, and the immunoreactivity was markedly increased 2 days after I–R. Five days after I–R, ubiquitin^+ 1 immunoreactivity was very weak in the SP. In all the IPC + ischemia-operated-groups, ubiquitin^+ 1 immunoreactivity in the SP was slightly decreased with time after I–R. Western blot analysis showed that, in all the IPC + ischemia-ischemia-groups, the levels of ubiquitin and ubiquitin^+ 1 proteins were well maintained after I–R. In brief, our findings suggest that the inhibition of the depletion of free ubiquitin and the formation of ubiquitin^+ 1 may have an essential role in inducing cerebral ischemic tolerance by IPC.     

Transient ischemia-induced change of CCR7 immunoreactivity in neurons and its new expression in astrocytes in the gerbil hippocampus

Chemokines and their receptors are important players in organism homeostasis, development and immune response to inflammatory stimuli. In the present study, we examined effects of ischemia-reperfusion injury on the immunoreactivity and protein levels of chemokine C–C motif receptor 7 (CCR7) in the gerbil hippocampus (CA1–3 regions) after 5 min of transient global cerebral ischemia. CCR7 immunoreactivity was dramatically changed in the pyramidal neurons of the CA1, not CA2/3, region after ischemia-reperfusion. The immunoreactivity was increased after ischemia-reperfusion, and it was barely found from 5 days post-ischemia. In addition, CCR7 immunoreactivity was newly expressed in astrocytes, not microglia, in the ischemic CA1 region from 5 days post-ischemia. However, we did not observe this finding in the ischemic CA2/3 region. Furthermore, CCR7 protein levels in the ischemic CA1 region were changed like the change pattern of its immunoreactivity. These results indicate that both CCR7 immunoreactivity and protein levels are distinctively altered only in the CA1 region after transient cerebral ischemia and that the changes in CCR7 expression may be related to the ischemia-induced delayed neuronal death.     

Facilitated recovery from postischemic suppression of protein synthesis in the gerbil brain with ischemic tolerance

Preconditioning of the gerbil brain with a 2-min period of sublethal ischemia followed by 4 days of reperfusion protects against neuronal damage following a subsequent 3-min period of ischemia, which normally destroys pyramidal neurons in the CAI region of the hippocampus. To clarify the role of protein synthesis in this ischemic tolerance phenomenon, we performed an autoradiographic analysis with [¹⁴C]leucine at 4 h, 24 h, and 48 h after 3 min of ischemia with and without preconditioning. General protein synthesis in the CAI region was severely suppressed after 4 h in both groups. The protein synthesis in CAI partially recovered after 24 h and fully recovered after 48 h in animals with preconditioning, but never recovered in animals without preconditioning. Protein synthesis in the neocortex and the striatum was suppressed in the early reperfusion periods only in animals without preconditioning. The results show that the ischemic tolerance is closely related to the facilitated recovery from suppressed protein synthesis in the brain after ischemia.     

Induction of NADPH-diaphorase activity in the hippocampus in a rat model of cerebral ischemia and ischemic tolerance

Preconditioning of the brain with sublethal ischemia induces tolerance to subsequent lethal periods of ischemia (ischemic tolerance). In this study, we used NADPH-diaphorase histochemistry to investigate the postischemic changes of nitric oxide synthase (NOS) in the hippocampus in a rat model of cerebral ischemia and ischemic tolerance. Forebrain ischemia was induced by 4-vessel occlusion for 3 min as an ischemic preconditioning. Three days after the preconditioning or sham operation, second ischemia was induced for 6 min. A transient increase in NADPH-diaphorase activity, beginning after 2 h and maximal after 1 day, was observed in CA1 pyramidal neurons of rats subjected to 3 min of preconditioning ischemia as well as 6 min of subsequent ischemia both with and without preconditioning. In addition, expression of NADPH-diaphorase activity was seen in reactive glial cells in the damaged CA1 region of animals subjected to 6 min of ischemia without preconditioning. Thus, direct involvement of increased NADPH-diaphorase activity in ischemic tolerance was not suggested because the increased NADPH-diaphorase activity preceded the induction of ischemic tolerance which takes place 1–7 days after preconditioning. However, the present findings suggest that the induction of neuronal NADPH-diaphorase activity occurs in response to cerebral ischemia.     

Induction of 27-kDa heat shock protein following cerebral ischemia in a rat model of ischemic tolerance

Preconditioning the brain with sublethal cerebral ischemia induces tolerance to subsequent lethal periods of ischemia (ischemic tolerance). The purpose of this study is to investigate the role of low-molecular weight stress proteins, 27-kDa heat shock protein (HSP27) and αB crystallin, in ischemic tolerance. We measured the content of these proteins with enzyme immunoassay in the rat hippocampus and cerebral cortex following 6 min of ischemia with and without preconditioning with 3 min of ischemia and 3 days of reperfusion. We also visualized the localization of HSP27 immunohistochemically in comparison with that of HSP70. A 3-min period of ischemia caused a 2.4-fold increase in HSP27 content in the hippocampus after 3 days. Immunohistochemical localization of HSP27 was found in glial cells in all subregions of the hippocampus, whereas HSP70 immunostaining was seen only in CA1 pyramidal neurons. HSP27 content in the hippocampus decreased 2 h after 6 min of ischemia. HSP27 content progressively increased in the unpreconditioned hippocampus after 1 and 3 days, but returned to preischemic levels in the preconditioned hippocampus. HSP27 and HSP70 immunostaining was seen in CA1 pyramidal neurons after 1 day both with and without preconditioning. After 3 and 7 days, an intense HSP27 staining was observed in reactive glial cells in the CA1 without preconditioning, whereas the staining decreased in the preconditioned hippocampus. HSP70 staining was seen only in neurons at these time points. We observed no significant changes in HSP27 content in the cerebral cortex although neurons in the third and fifth layers were immunostained after 1 and 3 days. We observed no alterations in αB crystallin content after ischemia both in the hippocampus and the cortex. The present study demonstrated that cerebral ischemia induces HSP27 expression but not αB crystallin. Both HSP27 and HSP70 induction had a good temporal correlation with the induction of ischemic tolerance. However, different sites of action were suggested because the localization and cell types of HSP27 induction were quite different from those of HSP70 induction. The result suggests that it is unlikely that HSP27 is directly involved in the protection afforded by ischemic preconditioning.     

Post-ischemic hypothermia for 24 h in P7 rats rescues hippocampal neuron: Association with decreased astrocyte activation and inflammatory cytokine expression

Hypothermia is an effective method for reducing the neuronal damage induced by hypoxia–ischemia (HI) but the underlying mechanism remains unclear. To investigate the effects of post-HI hypothermia on the developing brain, 7-day-old rats were subjected to left carotid artery ligation followed by 8% oxygen for 2 h. They were divided into a hypothermia group (rectal temperature 32–33 °C for 24 h) and a normothermia group (36–37 °C for 24 h) immediately after hypoxia–ischemia. Animals were sacrificed at 12, 24 and 72 h for gene analysis and 0, 1, 3 and 7 days for protein analysis after HI. There was a significant decrease in infarct volume in the hypothermia group at 7 days after HI compared with that in the normothermia group. The hypothermia group had more neuronal nuclei (NeuN) positive neurons and lower levels of glial fibrillary acidic protein (GFAP) mRNA and immunoreactivity in the hippocampus CA1 region than the normothermia group. Real-time PCR showed no significant difference in glial cell line-derived neurotrophic factor (GDNF) mRNA expression in the hippocampus in the two groups at various time points after HI. However, GDNF protein level was significantly increased in the hypothermia group. On the other hand, mRNA and protein levels of the inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) were dramatically decreased in the hypothermia compared with the normothermia group. The present findings highlight an apparent association between inhibition of hippocampal neuron loss by hypothermia and decreased astrocytosis and inflammatory cytokine release after hypoxia–ischemia in the developing brain.     

Evaluation of preconditioning treatments to protect near-pure cortical neuronal cultures from in vitro ischemia induced acute and delayed neuronal death

We evaluated the efficacy of cycloheximide, heat stress, NMDA receptor blockade (MK801/AP-5), oxygen--glucose deprivation, hypoxia, hypothermia and TNFalpha preconditioning to protect cortical neurons from in vitro ischemic insults that result in acute necrotic and delayed apoptotic neuronal death. Preconditioning treatments were performed 22--24 h before in vitro ischemia. In vitro ischemia was carried out in 96-well microtitre strip-plates by washing neuronal cultures with a balanced salt solution containing 25 mM 2-deoxy-D-glucose and incubating in an anaerobic chamber. Glutamate receptor blockers were present during in vitro ischemia to induce delayed neuronal death. Cycloheximide, heat stress, MK801 and oxygen--glucose deprivation preconditioning were neuroprotective in both acute and delayed in vitro ischemic neuronal death models. AP-5 preconditioning and a 12 h post-MK801 preconditioning interval protected neurons from acute ischemic neuronal death only. Hypoxia, TNFalpha and hypothermic preconditioning provided no neuronal protection in the in vitro ischemia models. This study has confirmed for the first time that several preconditioning treatments can protect neurons from in vitro ischemia induced acute necrotic and delayed apoptotic neuronal death. In addition, a unique feature of this study is the finding that preconditioning could be induced in near-pure primary cortical neuronal cultures, thus confirming that ischemic tolerance is an intrinsic property of neurons and provides a simplified culture system for identifying neuroprotective proteins.     

Neuronal immunoreactivity for mannose-binding lectin after venous occlusion-induced focal cerebral ischemia in rats

A recent research reveals that complement activation exacerbates cerebral infarction. However, involvement of the lectin pathway, (the third complement activation pathway) in cerebral ischemia is not well studied. In this study, we investigated the appearance of mannose-binding lectin (MBL) in ischemic brain tissue. Male Wistar rats (n = 25) were divided into three groups: untreated control, sham, and vein occlusion (VO). Rats in the VO group had two adjacent photochemically occluded cortical veins. Regional cerebral blood flow (rCBF) was measured in the sham and VO groups. Rats were perfusion-fixed at 72 h in the sham group and at 3, 24, and 72 h after inducing ischemia in the VO group. Neuronal immunoreactivity for MBL, C1q, C3, and C5b-9 was graded on a scale from 0 (no staining) to 4 (strong immunoreactivity). rCBF significantly decreased in the VO group compared to the sham group. Immunohistochemical staining results were negative in the control group. MBL immunoreactivity was significant increased at 24 and 72 h after inducing ischemia in the VO group compared to the sham group. After inducing ischemia, C1 immunoreactivity was significantly increased at 3, 24, and 72 h, while C3 immunoreactivity increased at 72 h. C5b-9 immunoreactivity exhibited a tendency for only positive staining. In brain tissue after focal cerebral ischemia, MBL expression, as well as C1q and C3, appeared at 3 h, peaked at 24 h, and was maintained at 72 h. These results suggest that an MBL inhibitor administered during the relatively early ischemic phase might attenuate tissue damage.     

Neuroprotective effects of ischemic preconditioning on hippocampal CA1 pyramidal neurons through maintaining calbindin D28k immunoreactivity following subsequent transient cerebral ischemia

Ischemic preconditioning elicited by a non-fatal brief occlusion of blood flow has been applied for an experimental therapeutic strategy against a subsequent fatal ischemic insult. In this study, we investi-gated the neuroprotective effects of ischemic preconditioning (2-minute transient cerebral ischemia) on calbindin D28k immunoreactivity in the gerbil hippocampal CA1 area following a subsequent fatal tran-sient ischemic insult (5-minute transient cerebral ischemia). A large number of pyramidal neurons in the hippocampal CA1 area died 4 days after 5-minute transient cerebral ischemia. Ischemic preconditioning reduced the death of pyramidal neurons in the hippocampal CA1 area. Calbindin D28k immunoreactivity was greatly attenuated at 2 days after 5-minute transient cerebral ischemia and it was hardly detected at 5 days post-ischemia. Ischemic preconditioning maintained calbindin D28k immunoreactivity after transient cerebral ischemia. These findings suggest that ischemic preconditioning can attenuate transient cerebral ischemia-caused damage to the pyramidal neurons in the hippocampal CA1 area through maintaining cal-bindin D28k immunoreactivity.     

Diabetes mellitus influences the expression of NPY and VEGF in neurons of rat trigeminal ganglion

BackgroundDiabetes mellitus (DM) influences the trigeminal nerve function by changing the pain response and transduction of the orofacial sensory pathways. It affects the inflammatory response via neuropeptide Y (NPY) and vascular endothelial growth factor (VEGF), which could potentially have a relevant role in the pathophysiology of diabetic neuropathy. The aim was to investigate expression of VEGF and NPY in subpopulations of trigeminal ganglion (TG) neurons in rat models of early DM1 and DM2.MethodsDM1 model was induced by an intraperitoneal (i.p.) injection of streptozotocin (STZ) (55 mg/kg). DM2 rats were fed with a high fat diet (HFD) for two weeks and then received 35 mg/kg of STZ i.p. Two weeks and 2 months after the STZ-diabetes induction, rats were sacrificed and TG was immunohistochemically analyzed for detection of VEGF and NPY expression, and also double immunofluorescence labeling with isolectin (IB4) was completed.ResultsAn increased percentage of NPY+ neurons was observed 2 weeks after DM1 and 2 months post DM2 induction. NPY immunoreactivity was restricted to IB4-negative small-diameter and IB4+ neurons. Two weeks post induction, DM1 rats showed an increased percentage of VEGF/IB4− large neurons and DM2 rats showed an increased percentage of VEGF/IB4+ neurons. Two months after DM induction, the DM1 group showed a reduced percentage of VEGF/IB4 − small neurons.ConclusionThe observed changes may play a critical role in the modulation of nociceptor activity and plasticity of primary sensory trigeminal neurons. The results contribute to the understanding of the basic pathophysiology of trigeminal diabetic neuropathy.     

Neuroprotective effects of neuropeptide Y-Y2 and Y5 receptor agonists in vitro and in vivo

It is generally assumed that neurodegeneration is connected with glutamatergic hyperactivity, and that neuropeptide Y (NPY) inhibits glutamate release. Some earlier studies indicated that NPY may have neuroprotective effect; however, the results obtained so far are still divergent, and the role of different Y receptors remains unclear. Therefore in the presented study we investigated the neuroprotective potential of NPY and its Y2, Y5 or Y1 receptor (R) ligands against the kainate (KA)-induced excitotoxicity in neuronal cultures in vitro, as well as in vivo after intrahippocampal KA injection and also in an ischemic middle cerebral artery occlusion model after intraventricular injection of Y2R agonist. NPY compounds were applicated 30 min, 1, 3 or 6 h after the start of the exposure to KA, or 30 min after the onset of ischemia. Our results indicate the neuroprotective activity of NPY and its Y2R and Y5R ligands against the kainate-induced excitotoxicity in primary cortical and hippocampal cultures. Importantly, NPY was effective when given as late as 6 h, while Y2R or Y5R agonists 3 h, after starting the exposure to KA. In in vitro studies those protective effects were inhibited by the respective receptor antagonists. Neuroprotection was also observed in vivo after intrahippocampal injection of Y2R and Y5R agonists 30 min or 1 h after KA. No protection was found either in vitro or in vivo after the Y1R agonist. The Y2R agonist also showed neuroprotective activity in the ischemic model. The obtained results indicate that neuropeptide Y produces neuroprotective effect via Y2 and Y5 receptors, and that the compounds may be effective after delayed application.     

Glutamate transporter type 3 mediates isoflurane preconditioning-induced acute phase of neuroprotection in mice

A pre-exposure to isoflurane reduces ischemic brain injury in rodents (isoflurane preconditioning). This neuroprotection has acute and delayed phases. Our previous in vitro studies suggest that the acute phase may involve excitatory amino acid transporters (EAATs). We determine whether this protection involves EAAT3, the major neuronal EAAT. Adult male EAAT3 knockout mice and their wild-type littermates were exposed or were not exposed to 1.5% isoflurane for 30 min. Sixty minutes later, they were subjected to a 90- or 60-min middle cerebral arterial occlusion (MCAO). Their neurological outcomes were evaluated 24 h after the MCAO. In another experiment, cerebral cortex was harvested for Western blotting at 30 min after animals were exposed to 1.5% isoflurane for 30 min. Here, we showed that isoflurane reduced brain infarct volumes and improved neurological functions of wild-type mice after a 90-min MCAO. However, isoflurane pre-exposure did not change the neurological outcome of EAAT3 knockout mice no matter whether the MCAO was for 90 min or 60 min. Isoflurane increased phospho-Akt, a survival-promoting protein, in the wild-type mice but not in the EAAT3 knockout mice. The isoflurane-induced neuroprotection in the wild-type mice was abolished by LY294004, an Akt activation inhibitor. LY294004 alone did not affect the neurological outcome of the wild-type or EAAT3 knockout mice after focal brain ischemia. These results suggest that the isoflurane preconditioning-induced acute phase of neuroprotection involves EAAT3. The downstream event includes Akt activation.     

A pilot study of sleep,cognition, and respiration under 4 weeks of intermittent nocturnal hypoxia in adult humans

Study objectivesA pilot study to examine the effects of intermittent nocturnal hypoxia on sleep, respiration and cognition in healthy adult humans.MethodsParticipants were eight healthy, non-smoking subjects (four male, four female), mean age of 26.4 ± 5.2 years, and BMI 22.3 ± 2.6 kg/m², exposed to 9 h of intermittent hypoxia between the hours of 10 P.M. and 7 A.M. for 28 consecutive nights. At a simulated altitude of 13,000 feet (FIO₂ 0.13), intermittent hypoxia was achieved by administering nasal nitrogen, alternating with brief (approximately 5 s) boluses of nasal oxygen. Pre- and post-exposure assessments included polysomnography, attention (20-min Psychomotor Vigilance Test), working memory (10-min verbal 2 and 3-back), Multiple Sleep Latency Test, and the Rey Auditory Verbal Learning Test. Obstructive and non-obstructive respiratory events were scored.ResultsOverall sleep quality showed worsening trends but no statistically significant change following exposure. There was no difference after hypoxia in sleepiness, encoding, attention or working memory. Hyperoxic central apneas and post-hyperoxic respiratory instability were noted as special features of disturbed respiratory control induced by intermittent nocturnal hypoxia.ConclusionsIn this model, exposure to nocturnal intermittent hypoxia for 4 weeks caused no significant deficits in subjective or objective alertness, vigilance, or working memory.     

Cerebral ischemic preconditioning induces lasting effects on CA1 neuronal survival, prevents memory impairments but not ischemia-induced hyperactivity

In ischemic preconditioning, prior exposure to a short 3-min global ischemia provides substantial protection against the deleterious effects of a subsequent prolonged ischemic insult in rats. The objective of the present study was to determine if the neuronal protection induced by ischemic preconditioning influence functional recovery following a 6-min ischemic insult in rats. Animals received either sham-operation, a 3-min ischemia, a preconditioning 3-min global ischemia followed 3 days later by a 6-min global ischemia or a single 6-min global ischemia. Open field habituation, memory performance in the 8-arm radial maze and object recognition were assessed at different intervals following ischemia. Our findings revealed that preconditioning reversed ischemia-induced spatial memory deficits in the 8-arm radial maze, as suggested by significant reduction of working memory errors in preconditioned as compared to ischemic animals. Preconditioning also attenuated ischemia-induced object recognition deficits at short-term intervals. Nonetheless, preconditioning failed to alter ischemia-induced hyperactivity as demonstrated by enhanced behavioral activity in the open field in both preconditioned and ischemic animals compared to 3-min ischemic and sham-operated rats. CA1 cell counts revealed significant neuronal sparing in preconditioned animals that was observed 6-month following reperfusion. Together, these findings suggest that neuronal survival in preconditioned rats is associated with significant improvements of hippocampal-dependent memory functions and, further support that ischemia-induced hyperactivity may not solely depend on selective neuronal damage to hippocampal neurons.     

Chronic intermittent hypoxia reduces neurokinin-1 (NK1) receptor density in small dendrites of non-catecholaminergic neurons in mouse nucleus tractus solitarius

Chronic intermittent hypoxia (CIH) is a frequent concomitant of sleep apnea, which can increase sympathetic nerve activity through mechanisms involving chemoreceptor inputs to the commissural nucleus of the solitary tract (cNTS). These chemosensory inputs co-store glutamate and substance P (SP), an endogenous ligand for neurokinin-1 (NK₁) receptors. Acute hypoxia results in internalization of NK₁ receptors, suggesting that CIH also may affect the subcellular distribution of NK₁ receptors in subpopulations of cNTS neurons, some of which may express tyrosine hydroxylase, the rate-limiting enzyme for catecholamine synthesis (TH). To test this hypothesis, we examined dual immunolabeling for the NK₁ receptor and TH in the cNTS of male mice subjected to 10 days or 35 days of CIH or intermittent air. Electron microscopy revealed that NK₁ receptors and TH were almost exclusively localized within separate somatodendritic profiles in cNTS of control mice. In dendrites, immunogold particles identifying NK₁ receptors were prevalent in the cytoplasm and on the plasmalemmal surface. Compared with controls, CIH produced a significant region-specific decrease in the cytoplasmic (10 and 35 days, P < 0.05, unpaired Student t-test) and extrasynaptic plasmalemmal (35 days, P < 0.01, unpaired Student t-test) density of NK₁ immunogold particles exclusively in small (< 0.1 µm) dendrites without TH immunoreactivity. These results suggest that CIH produces a duration-dependent reduction in the availability of NK₁ receptors preferentially in small dendrites of non-catecholaminergic neurons in the cNTS. The implications of our findings are discussed with respect to their potential involvement in the slowly developing hypertension seen in sleep apnea patients.     

Preserved neurotransmitter receptor binding following ischemia in preconditioned gerbil brain

Preconditioning the brain with sublethal ischemia induces tolerance to subsequent ischemic insult. Using [3H]quinuclidinyl benzilate (QNB), [3H]MK 801, [3H]cyclohexyladenosine, [3H]muscimol, and [3H]PN200-110, we investigated the alterations in neurotransmitter receptor and calcium channel binding in the gerbil hippocampus following ischemia with or without preconditioning. Two-minute forebrain ischemia, which produced no neuronal damage, resulted in no alterations in binding except for a slight reduction in [3H]QNB binding in the CA1 subfield. Three-minute ischemia destroyed the majority of CA1 pyramidal cells and caused, in CA1, reductions in binding of all ligands used. Preconditioning with 2-min ischemia followed by 4 days of reperfusion protected against CA1 neuronal damage and prevented the reductions in binding although [3H]QNB and [3H]PN200-110 binding transiently decreased in the early reperfusion period, suggesting down-regulation. Thus, preconditioning protects against damage to the neurotransmission system as well as histopathological neuronal death.     

Protective action of tetramethylpyrazine on the medulla oblongata in rats with chronic hypoxia

Tetramethylpyrazine (TMP), one of the active ingredients of the Chinese herb Lingusticum Wallichii Frantchat (Chuan Xiong), plays an important role in neuroprotection. However, the protective effect of TMP on the medulla oblongata, the most important region of the brain for cardiovascular and respiratory control, during chronic hypoxia remains unclear. In this study, we examined the neuroprotective effect of TMP on the medulla oblongata after chronic hypoxic injury in rats. Male Sprague–Dawley rats were randomly divided into four groups: control group, TMP group, chronic hypoxia group, and chronic hypoxia + TMP group. Rats were exposed to hypoxia (10% (v/v) O₂) or normoxia for 6 h daily for 14 days. TMP (80 mg/kg) or vehicle (saline) was injected intraperitoneally 30 min before experimentation. Loss of neurons in the pre-Bötzinger complex, the nucleus ambiguus, the nucleus tractus solitarius, the hypoglossal nucleus and the facial nucleus were evaluated by Nissl staining. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were measured, and apoptosis was monitored using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. The level of Bcl-2 mRNA and Bax mRNA was quantitatively measured by RT-PCR analysis. TMP protected Nissl bodies of neurons from injury in all nuclei observed, and reduced the loss of neurons in the nucleus ambiguus, the nucleus tractus solitarius, and the hypoglossal nucleus in rats subjected to chronic hypoxia. TMP upregulated SOD activity and inhibited the increase in MDA content in the medulla oblongata of hypoxic rats. In addition, TMP decreased the rate of apoptosis index (the percentage of apoptotic cells against the total number of cells) in all medullary structures examined, excepting the nucleus ambiguus and inhibited the decrease in Bcl-2 mRNA levels in the medulla oblongata following hypoxia. Our findings indicate that TMP may protect the medullary structures that are involved in cardiovascular and respiratory control from injury induced by chronic hypoxia in rats via its anti-oxidant and anti-apoptotic effects.     

Neuronal relationship between orexin-A- and neuropeptide Y-induced orexigenic actions in goldfish

Orexin-induced orexigenic action is mediated by neuropeptide Y (NPY) in goldfish and rodents. A previous study indicated that NPY-induced orexigenic action may also be mediated by orexin-A in goldfish. However, there is little information about the mutual actions of orexin-A and NPY in the goldfish. Therefore, using their specific receptor antagonists, we examined whether the orexigenic actions of orexin-A and NPY mutually interact in the goldfish. The stimulatory effect of intracerebroventricular injection of NPY at 1 pmol/g body weight (BW) on food intake was abolished by treatment with the orexin receptor-1 antagonist, SB334867, at 10 pmol/g BW whereas the NPY Y1-receptor antagonist, BIBP3226, at 100 pmol/g BW attenuated orexin-A (at 2.8 pmol/g BW)-stimulated feeding. This led us, using a double-immunostaining method and confocal laser scanning microscopy, to investigate whether orexin-A- and NPY-containing neurons in the goldfish brain have direct mutual inputs. Orexin-A- and NPY-like immunoreactivities were distributed throughout the brain, especially in the diencephalon. Orexin-A- and NPY-containing neurons were located in a region of the hypothalamus, the nucleus posterioris periventricularis (NPPv), in close proximity to each other: NPY-containing nerve fibers or endings lay in close apposition to orexin-A-containing neurons in the NPPv, and orexin-A-containing nerve fibers or endings also lay in close apposition to NPY-containing neurons in the same region. These results indicate that, in goldfish, orexin-A- and NPY-induced orexigenic actions are mediated by mutual signaling pathways.     

Cerebellar abnormalities following hypoxia alone compared to hypoxic–ischemic forebrain injury in the developing rat brain

Two-day-old (P2) rat pups were subjected to either a global hypoxia or to electrocoagulation of the right carotid artery followed by 2.5 h hypoxia. Cellular and regional injury in the cerebellum (CB) was studied at 1, 2 and 19 days using immunohistology. Following hypoxia and hypoxia–ischemia, all neuronal populations of the CB were damaged in a subset of Purkinje cells. The decrease in the number of interneurons, as well as the thickness of molecular and granular layers was significant following hypoxia. Diffuse white matter damage, with loss of preoligodendrocytes was more severe following hypoxia than hypoxia–ischemia. Global hypoxia in the rat at P2 produces extensive damage to many cell types in different areas of the CB. The addition of unilateral forebrain ischemia does not increase the severity of these changes. Our data provide insight into the mechanisms of the changes observed in the CB of premature newborns.     

Ischemic preconditioning upregulates expression of phospholipase D2 in the rat hippocampus

To investigate the possible involvement of phospholipase D2 (PLD2) in the induction of ischemic tolerance, we analyzed the distribution and time course of PLD2 expression in the rat hippocampus after a sublethal period of ischemia. Forebrain ischemia was induced by four-vessel occlusion for 3 min. Increased PLD2 immunoreactivity after this sublethal ischemia was observed in CA1 pyramidal neurons of the rat hippocampus. In tolerance-acquired CA1 neurons, PLD2 immunoreactivity was upregulated as early as 12 h post-ischemia and was most prominent at 1–3 days, with expression sustained for at least 7 days, as shown by a time course of immunoblotting and measurement of the enzymatic activity of PLD. PLD2 expression was also increased in ischemia-resistant CA3 neurons and dentate granule cells, although weaker staining intensity was noted. Further, we showed that, in cultured SK-N-BE(2)C human neuroblastoma cells, overexpression of PLD2 inhibited cell death by chemical hypoxia induced with potassium cyanide and deoxyglucose. These data suggest that upregulation of PLD2 might be involved in the neuroprotective mechanism of ischemic tolerance in the rat hippocampus. This research was supported by a grant (M103KV010010-06K2201-01010) from Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, the Republic of Korea.