Role of the hippocampal CA2 region following postischemic hypothermia in gerbil

To investigate the changes in the principal subunit of N-methyl-D-aspartate (NMDA) receptor 1 (NR1) following the transient ischemia and postischemic hypothermia, in situ hybridization was used in the gerbil hippocampus. One of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, Glutamate receptor 2 (GluR2) was also investigated to compare with NR1. Even at 1 day, NR1 and GluR2 mRNAs in the CA1 region were reduced following ischemia. Although postischemic hypothermia prevented almost all the neuronal cell death by ischemia and inhibited the reduction of NR1 and GluR2 mRNAs in the CA1 region after 7 days, the downregulation of NR1 mRNA in the CA2 region was observed even at 1 day. This change was specific for NR1 and not for GluR2. These results suggest that the changes in NR1 and GluR2 receptors at the mRNA level would occur in spite of postischemic hypothermia. The phenomenon in the CA2 region may play an important role to rescue neuronal cell death by ischemia.     

Transgenic rescue of SNAP-25 restores dopamine-modulated synaptic transmission in the coloboma mutant

Previous cerebral ischemia studies have reported the limitations of restricted periods of postischemic hypothermia in producing long-term neuroprotection. The present experiment attempts to determine whether delayed treatment with the free radical scavenger N-tert-butyl-a-phenylnitrone (PBN) is protective at 2 months following transient global forebrain ischemia, and whether additive effects can be observed when PBN is administered in combination with moderate hypothermia. For this aim rats were subjected to 10 min of two-vessel forebrain ischemia followed by (a) 3 h of postischemic normothermia (37 degrees C); (b) 3 h of postischemic hypothermia (30 degrees C); (c) normothermic procedures combined with delayed injections of PBN (100 mg/kg) on days 3, 5 and 7 post-insult; (d) postischemic hypothermia combined with delayed PBN treatment; or (e) sham procedures. Outcome measures included cognitive behavioral testing and quantitative histopathological analysis at 2 months. Postischemic PBN injections induced a systemic hypothermia (1.5 degrees C-2.0 degrees C) that lasted for 2-2.5 h. Water maze testing revealed significant performance deficits relative to shams in the normothermic ischemic group, with the postischemic hypothermia and PBN groups showing intermediate values. A significant attenuation of cognitive deficits was observed in the animal group receiving the combination postischemic hypothermia and delayed PBN treatment. Quantitative CA1 hippocampal cell counts indicated that each of the ischemia groups exhibited significantly fewer viable CA1 neurons compared to sham controls. However, in rats receiving either delayed PBN treatment or 3 h of postischemic hypothermia, significant sparing of CA1 neurons relative to the normothermic ischemia group was observed. These data indicate that hypothermia combined with PBN treatment provides long-term cognitive improvement compared to nontreatment groups. PBN-induced mild hypothermia could contribute to the neuroprotective effects of this pharmacological strategy.     

Effects of combined postischemic hypothermia and delayed N-tert-butyl-a-pheylnitrone (PBN) administration on histopathological and behavioral deficits associated with transient global ischemia in rats

Previous cerebral ischemia studies have reported the limitations of restricted periods of postischemic hypothermia in producing long-term neuroprotection. The present experiment attempts to determine whether delayed treatment with the free radical scavenger N-tert-butyl-a-phenylnitrone (PBN) is protective at 2 months following transient global forebrain ischemia, and whether additive effects can be observed when PBN is administered in combination with moderate hypothermia. For this aim rats were subjected to 10 min of two-vessel forebrain ischemia followed by (a) 3 h of postischemic normothermia (37°C); (b) 3 h of postischemic hypothermia (30°C); (c) normothermic procedures combined with delayed injections of PBN (100 mg/kg) on days 3, 5 and 7 post-insult; (d) postischemic hypothermia combined with delayed PBN treatment; or (e) sham procedures. Outcome measures included cognitive behavioral testing and quantitative histopathological analysis at 2 months. Postischemic PBN injections induced a systemic hypothermia (1.5°C–2.0°C) that lasted for 2–2.5 h. Water maze testing revealed significant performance deficits relative to shams in the normothermic ischemic group, with the postischemic hypothermia and PBN groups showing intermediate values. A significant attenuation of cognitive deficits was observed in the animal group receiving the combination postischemic hypothermia and delayed PBN treatment. Quantitative CA1 hippocampal cell counts indicated that each of the ischemia groups exhibited significantly fewer viable CA1 neurons compared to sham controls. However, in rats receiving either delayed PBN treatment or 3 h of postischemic hypothermia, significant sparing of CA1 neurons relative to the normothermic ischemia group was observed. These data indicate that hypothermia combined with PBN treatment provides long-term cognitive improvement compared to nontreatment groups. PBN-induced mild hypothermia could contribute to the neuroprotective effects of this pharmacological strategy.     

Protective effects of brief intra- and delayed postischemic hypothermia in a transient focal ischemia model in the neonatal rat

Hypothermia provides neuroprotection in virtually all animal models of ischemia, including adult stroke models and the neonatal hypoxic-ischemic (HI) model. In these studies, brief periods of hypothermia are examined in a neonatal model employing transient focal ischemia in a 7-day-old rat pup. Pups underwent permanent middle cerebral artery (MCA) occlusion coupled with a temporary (1 h) occlusion of the ipsilateral common carotid artery (CCA). This study included five treatment groups: (1) normothermic (Normo)-brain temperature was maintained at 37 degrees C; (2) intraischemic hypothermia (IntraH)-28 degrees C during the 1-h ischemic period only; (3) postischemic hypothermia (PostH)-28 degrees C for the second hour of reperfusion only; (4) late-onset postischemic hypothermia (LPostH) cooled to 28 degrees C for the fifth and sixth hours of reperfusion only; and (5) Shams. After various times (3 days-6 weeks), the lesion was assessed using 2,3,5-triphenyltetrazolium chloride (TTC) or hematoxylin and eosin (H&E) stains. Intraischemic hypothermia resulted in significant protection in terms of survival, lesion size, and histology. Postischemic hypothermia was not effective in reducing lesion size early after ischemia, but significantly reduced the eventual long-term damage (2-6 weeks). Late-onset postischemic hypothermia did not reduce infarct volume. Therefore, both intraischemic and postischemic hypothermia provided neuroprotection in the neonatal rat, but with different effects on the degenerative time course. While there were no observable differences in simple behaviors or growth, all hypothermic conditions significantly reduced mortality rates. While the protection resulting from intraischemic hypothermia is similar to what is observed in other models, the degree of long-term ischemic protection observed after 1 h of postischemic hypothermia was remarkable and distinct from what has been observed in other adult or neonatal models.     

Postischemic Hypothermia and IL-10 Treatment Provide Long-Lasting Neuroprotection of CA1 Hippocampus Following Transient Global Ischemia in Rats

Experimental studies have demonstrated that postischemic therapeutic interventions may delay rather than provide long-lasting neuroprotection. The purpose of this study was to determine whether mild hypothermia (33-34 degrees C) combined with the anti-inflammatory cytokine interleukin-10 (IL-10) would protect the CA1 hippocampus 2 months after ischemia. Rats were subjected to 12.5 min of normothermic (37 degrees C) forebrain ischemia by two-vessel occlusion followed immediately by: (a) 4 h of normothermic (37 degrees C) reperfusion (n = 5); (b) 4 h of postischemic hypothermia (33-34 degrees C) (n = 5); (c) 4 h of normothermia plus IL-10 (5 micrograms) treatment 30 min after ischemia and at 3 days (n = 5); or (d) 4 h of hypothermia plus IL-10 treatment (n = 5). Rats survived for 2 months and were perfusion fixed for quantitative histopathological assessment of CA1 hippocampus. Postischemic normothermia and hypothermia, as well as normothermia plus IL-10 treatment led to severe damage of the CA1 hippocampus. In contrast, the combined treatment of hypothermia with IL-10 treatment improved overall neuronal survival by 49% compared to normothermic ischemia (P < 0.01). These data emphasize the detrimental consequences of secondary inflammatory responses on ischemic neuronal damage after transient global ischemia. In postinjury settings where restricted durations of mild hypothermia can be induced, anti-inflammatory treatments, including IL-10, may promote chronic neuroprotection.     

Neuroprotection role of adenosine under hypothermia in the rat global ischemia involves inhibition of not dopamine release but delayed postischemic hypoperfusion

Adenosine (ADO) has an important role in the ischemic brain as an endogenous neuroprotective factor. On the other hand, intraischemic hypothermia ameliorates ischemic neuronal injury. To investigate the effect of ADO during intraischemic mild hypothermia, the extracellular concentration of ADO, its metabolites, dopamine (DA), and local cerebral blood flow were measured in rat striatum during and after 20 min of global ischemia. Additionally, the histopathological outcome was estimated after 48 h of recirculation. Three experimental groups were used: (1) a normothermic group (NT) maintained at 37 degrees C during and after ischemia; (2) a hypothermic group (HT), exposed to intraischemic hypothermia (32.0 degrees C) and postischemic normothermia; and (3) a hypothermia plus theophylline group (HT+T), with the same temperature conditions as in the HT group, combined with intravenously administration of theophylline (10 mg/kg), an antagonist of adenosine receptor, which was given 10 min before ischemia. The level of ADO in HT was significantly higher than ADO levels in NT. In contrast, ischemic DA release was significantly inhibited in HT compared with NT. Theophylline administration had no effect on intraischemic hypothermia induced modulation of extracellular ADO and DA concentration. The postischemic delayed hypoperfusion was ameliorated in HT, and theophylline eliminated this effect in HT+T. A protective effect on histopathological outcome was observed in HT and HT+T. These results suggest that ADO plays an essential role in the inhibition of postischemic delayed hypoperfusion, but this effect is not crucial role in the protective effect induced by intraischemic hypothermia.     

Prior deafferentation confers long term protection to CA1 against transient forebrain ischemia and sustains GluR2 expression

Hippocampal CA1 pyramidal neurons undergo delayed neurodegeneration after transient forebrain ischemia, and the phenomenon is dependent upon hyperactivation of l-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype of glutamate receptors, resulting in aberrant intracellular calcium influx. The GluR2 subunit of AMPA receptors is critical in limiting the influx of calcium. The CA1 pyramidal neurons are very sensitive to ischemic damage and attempts to achieve neuroprotection, mediated by drugs, have been unsuccessful. Moreover, receptor antagonism strategies in the past have failed to provide long-term protection against ischemic injury. Long-term protection against severe forebrain ischemia can be conferred by fimbria-fornix (FF) deafferentation, which interrupts the afferent input to CA1. Our study evaluated the long-term protective effect of FF deafferentation, 12 days prior to induction of ischemia, on vulnerable CA1 neurons. Our results indicate that at 7 and 28 days post-ischemia, prior FF deafferentation protected 60% of neurons against ischemic cell death. Furthermore, we sought to evaluate whether FF deafferentation also sustained GluR2 levels in these neurons. GluR2 protein and mRNA expression were sustained by deafferentation at 70% of control following ischemia. Correlation studies revealed a positive correlation between GluR2 protein and mRNA level. These results demonstrate that protection conferred by FF deafferentation was long-term and related to sustained GluR2 expression.     

Disruption of the GluR2-NSF interaction protects primary hippocampal neurons from ischemic stress

A specific interaction between the AMPA receptor subunits GluR2 and GluR3 and the fusion protein NSF has recently been identified. Disruption of this interaction by adenoviral-mediated expression of a peptide (pep2m) corresponding to the NSF-binding region of GluR2 results in a dramatic reduction in surface expression of AMPA receptors in primary hippocampal neurons. Here we report that expression of pep2m from a recently developed neuronal-specific adenoviral system gave significant neuroprotection to primary CA1-CA3 hippocampal neurons following stimulation with kainate (KA) and this was accompanied by a reduction in Ca(2+) influx. Protection was also observed following glucose deprivation and exposure to ischemic buffer in the absence of any NMDA receptor antagonists. These results provide strong evidence that AMPA receptors play a direct role in mediating postischemic neurotoxicity.     

Regionally selective effects of NMDA receptor antagonists against ischemic brain damage in the gerbil

This study compared the ability of three N-methyl-D-aspartate (NMDA) receptor antagonists to prevent neuronal degeneration in an animal model of global cerebral ischemia. The model employed is characterized by damage to the striatum, hippocampus, and neocortex. Antagonists were administered to gerbils either before or after a 5-min bilateral carotid occlusion. The intraischemic rectal temperature was either maintained at 36-37 degrees C or allowed to fall passively to 28-32 degrees C. Antagonists and doses tested were 1 and 10 mg/kg of MK-801 (pre- or postischemia), 30 mg/kg of CGS 19755 preischemia, four 25 mg/kg doses of CGS 19755 administered between 0.5 and 6.5 h postischemia, and 40 mg/kg of MDL 27,266 (pre- or postischemia). All three NMDA receptor antagonists exhibited some degree of neuroprotective activity when the carotid occlusion was performed under normothermic conditions. Most of the treatments with antagonist markedly reduced striatal damage. CA1 hippocampal and neocortical pyramidal cells were spared by only three of the treatments, however, and the extent of neuroprotection varied widely from case to case. Toxic doses of antagonist were required to protect CA1 pyramidal cells from ischemic damage. Ischemic damage to hippocampal areas CA2-CA3a and CA4 appeared to be resistant to all of these treatments. Most CA1 pyramidal cells that were protected from degeneration by an NMDA receptor antagonist were histologically abnormal. The neuroprotective effects of MK-801 and intraischemic hypothermia appeared to be additive. MK-801 (10 mg/kg) consistently reduced the postischemic brain temperature, but only the magnitude of hypothermia produced soon after reperfusion correlated with its neuroprotective action. These results suggest that NMDA receptor antagonists are relatively poor neuroprotective agents against a moderately severe ischemic insult.     

AMPA receptor protein expression and function in astrocytes cultured from hippocampus.

Glutamate receptors guide the proliferation, migration, and differentiation of glial cells. Here, we characterize AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) and NMDA receptor protein expression and function and mRNA expression in hippocampal glial cultures. By immunocytochemistry, GluR2 (the subunit that limits the Ca(2+) permeability of AMPA receptors) exhibited prominent labeling in hippocampal glial cultures. Double-labeling of GluR2 with GFAP and with A2B5 revealed GluR2 subunit expression on type-1 and type-2 astrocyte lineage cells. GluR1 subunit expression was more prominent in type-1 than in type-2 astrocytes. To characterize functional properties of glutamate receptors expressed in cultured hippocampal astrocytes, we performed whole-cell patch clamp recording. Application of L-glutamate, AMPA, and kainate, but not NMDA, to small, rounded cells (morphologically identified as type-2 astrocytes) elicited inward currents which were blocked by the AMPA/kainate antagonist 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX). Cyclothiazide potentiated AMPA- and kainate-elicited currents, indicative of AMPA-preferring receptors. Current voltage analysis indicated that type-2 astrocyte AMPA receptors were electrically linear, indicative of GluR2-containing, Ca(2+)-impermeable AMPA receptors. By Northern blot analysis, GluR1 mRNA was highest in astrocyte cultures from cerebellum and hippocampus and moderate in astrocyte cultures from neocortex and striatum. GluR3 mRNA was detectable in astrocyte cultures from cerebellum and neocortex. GluR2 and NR1 mRNA expression were not detected in astrocytes cultured from any brain region examined. In situ hybridization studies showed wide expression of GluR1 mRNA in cultured astrocytes; GluR2 and GluR3 mRNAs were near background levels. Thus, cultured type-2 astrocytes express functional AMPA receptors in a cell-specific and region-specific manner, consistent with their role in neuronal-glial communication.     

Lithium ion does not protect brain against transient ischemia in gerbils.

It has been proposed that lithium ion desensitizes neuronal receptors that function via the inositol phospholipid signaling mechanism. We examined the effects of lithium chloride on the morphologic outcome after 5 minutes of cerebral ischemia induced in gerbils by occluding both common carotid arteries under brief halothane anesthesia. In three treated groups of 10 gerbils each, 5 meq/kg i.p. lithium chloride was given 2 days, 1 day, and 2 hours before ischemia; 2 hours before ischemia; or immediately after the end of ischemia. Corresponding control groups of nine or 10 gerbils each received equivalent volumes of saline injected at comparable times. All gerbils were perfusion-fixed 1 week later, and neuronal density of the hippocampal CA1 pyramidal cells was determined. Lithium induced very mild intraischemic systemic hypothermia, but postischemic hyperthermia developed in both treated and control groups. Neuronal densities were equal in corresponding groups. The results indicate that our regimen of lithium administration provides no benefit in survival of hippocampal neurons, and intraischemic hypothermia of less than 0.8 degrees C is not protective. Other strategies to inactivate the signal transduction system that is specific for excitatory neurotransmission should be evaluated.     

Post-ischemic diazepam does not reduce hippocampal CA1 injury and does not improve hypothermic neuroprotection after forebrain ischemia in gerbils

The hippocampal CA1 sector is especially vulnerable to brief forebrain ischemia. Excitotoxicity is widely thought to contribute to this cell death. Accordingly, drugs that presumably counteract excitotoxicity, such as GABAergic agonists, have been repeatedly tested and found to reduce CA1 cell loss. Post-ischemic diazepam reduces CA1 injury. However, diazepam also causes hypothermia, which by itself is neuroprotective. Most studies fail to adequately control for this confound. In this study, we tested whether diazepam reduces injury in temperature controlled gerbils subjected to brief forebrain ischemia. Furthermore, we tested whether diazepam augments hypothermic neuroprotection. All gerbils were implanted with a core temperature telemetry probe and a cannula for the subsequent insertion of a thermocouple probe to measure ischemic brain temperature. Subsequently, they were given a 5-min normothermic ischemic insult. In Experiment 1, two groups of gerbils were given 10 mg/kg doses of diazepam (i.p.) at both 30 and 90 min post-ischemia. Temperature was maintained in one group by heating lamps. Another group was administered saline. Diazepam reduced cell death at 7 days post-ischemia when the drug-induced hypothermia was permitted, but not when it was prevented. In Experiment 2, four groups of ischemic gerbils were treated starting at 12 h post-ischemia with prolonged hypothermia, diazepam and the combination or saline treatment. Hypothermia, but not diazepam, provided partial neuroprotection and diazepam did not augment hypothermic neuroprotection. Thus, neuroprotection with diazepam is solely due to hypothermia. These data do not support the clinical use of diazepam as a neuroprotectant after global ischemia.     

Regional and time‐dependent neuroprotective effect of hypothermia following oxygen‐glucose deprivation

The neuroprotective effect of hypothermia has been demonstrated in in vivo and in vitro models of cerebral ischemia. In regard to the hippocampus, previous studies have mainly focused on CA1 pyramidal neurons, which are very vulnerable to ischemia. But the dentate gyrus (DG), in which neuronal proliferation occurs, can also be damaged by ischemia. In this study, we explored the neuroprotective effect of postischemic hypothermia in different areas of the hippocampus after mild or severe ischemia. Organotypic hippocampal slice cultures were prepared from 6‐ to 8‐day‐old rats and maintained for 12 days. Cultures were exposed to 25 or 35 min of oxygen and glucose deprivation (OGD). Neuronal damage was quantified after 6, 24, 48, and 72 h by propidium iodide fluorescence. Mild hypothermia (33°C) was induced 1 h after the end of OGD and was maintained for a period of 24 h. Short OGD produced delayed neuronal damage in the CA1 area and in the DG and to a lesser extend in the CA3 area. Damage in CA1 pyramidal cells was totally prevented by hypothermia whereas neuroprotection was limited in the DG. Thirty‐five‐minute OGD induced more rapid and more severe cell death in the three regions. In this case, hypothermia induced 1 h after OGD was unable to protect CA1 pyramidal cells whereas hypothermia induced during OGD was able to prevent cell loss. This study provides evidence that neuroprotection by hypothermia is limited to specific areas and depends on the severity of the ischemia. © 2014 Wiley Periodicals, Inc.     

Postischemic neuroprotection in the ischemia-tolerant state gerbil hippocampus is associated with increased ligand binding to inhibitory GABA<Subscript>A</Subscript> receptors

Excitotoxic activation of glutamate receptors is thought to play a key role in delayed neuronal death (DND) of highly vulnerable hippocampal CA1 neurons after transient global ischemia. DND can be prevented by a short sublethal preconditioning (PC) stimulus. Recently, we demonstrated that ischemic PC, but not a single period of 5-min ischemia elicits a transient up-regulation of hippocampal [(3)H]muscimol binding to GABA(A) receptors. This indicates that activation of the GABAergic system may participate in the acquisition of neuroprotection. The present study was designed to test whether postischemic modulation of receptor binding also occurs in the ischemia-tolerant state, i.e., after a PC stimulus of 2.5-min ischemia and a subsequent normally lethal period of 5-min ischemia 4 days apart. Using receptor autoradiography, [(3)H]AMPA and [(3)H]muscimol binding to excitatory AMPA and inhibitory GABA(A) receptors was analyzed in hippocampal subfields CA1, CA3 and dentate gyrus at recirculation intervals of 30 min, 8, 24, 48, 96 h and 3 weeks. Postischemic hippocampal ligand binding to AMPA receptors remained unchanged at any time point investigated, but [(3)H]muscimol binding to GABA(A) receptors in CA1 neurons rendered tolerant to ischemia was up-regulated between 30 min and 48 h of recirculation. Our data suggest that a relative shift between excitatory and inhibitory neurotransmission may promote postischemic survival of CA1 neurons.     

Prolonged therapeutic hypothermia does not adversely impact neuroplasticity after global ischemia in rats

Hypothermia improves clinical outcome after cardiac arrest in adults. Animal data show that a day or more of cooling optimally reduces edema and tissue injury after cerebral ischemia, especially after longer intervention delays. Lengthy treatments, however, may inhibit repair processes (e.g., synaptogenesis). Thus, we evaluated whether unilateral brain hypothermia (∼33°C) affects neuroplasticity in the rat 2-vessel occlusion model. In the first experiment, we cooled starting 1 hour after ischemia for 2, 4, or 7 days. Another group was cooled for 2 days starting 48 hours after ischemia. One group remained normothermic throughout. All hypothermia treatments started 1 hour after ischemia equally reduced hippocampal CA1 injury in the cooled hemisphere compared with the normothermic side and the normothermic group. Cooling only on days 3 and 4 was not beneficial. Importantly, no treatment influenced neurogenesis (Ki67/Doublecortin (DCX) staining), synapse formation (synaptophysin), or brain-derived neurotropic factor (BDNF) immunohistochemistry. A second experiment confirmed that BDNF levels (ELISA) were equivalent in normothermic and 7-day cooled rats. Last, we measured zinc (Zn), which is important in plasticity, with X-ray fluorescence imaging in normothermic and 7-day cooled rats. Hypothermia did not alter the postischemic distribution of Zn within the hippocampus. In summary, cooling significantly mitigates injury without compromising neuroplasticity.     

亚低温对大鼠短暂全脑缺血后神经元凋亡的影响

目的 探讨亚低温对大鼠脑缺血后神经元凋亡的影响，揭示亚低温的部分神经保护机制。方法 采用“双侧颈总动脉阻断＋全身低血压”方法来建立大鼠短暂性全脑缺血模型。用神经元尼氏体亚甲兰特殊染色法观察大鼠脑缺血后海马CA1区神经元损害情况；原位细胞凋亡检测法（TUNEL染色）及电镜观察脑缺血后CA1区神经元凋亡情况。结果 与假手术组、低温缺血组相比，常温缺血组海马CA1区神经元缺失明显（P＜0．01）。常温及低温缺血组海马CA1区均存在神经元凋亡，但低温缺血组海马CA1区凋亡神经元数明显少于缺血组（P＜0．01）。结论 经“双侧颈总动脉阻断＋全身低血压”方法建立的大鼠短暂全脑缺血模型证实了亚低温的脑保护作用。全脑缺血后的迟发性神经元死亡很可能经由凋亡途径，而亚低温可通过抑制缺血性神经元凋亡而发挥一定的神经保护作用。     

Selective reduction of GluR2 protein in adult hippocampal CA3 neurons following status epilepticus but prior to cell loss

Kainic acid (KA) induces status epilepticus and delayed neurodegeneration of CA3 hippocampal neurons. Downregulation of glutamate receptor 2 (GluR2) subunit mRNA [the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) subunit that limits Ca²⁺ permeability] is thought to a play role in this neurodegeneration, possibly by increased formation of Ca²⁺ permeable AMPA receptors. The present study examined early hippocampal decreases in GluR2 mRNA and protein following kainate-induced status epilepticus and correlated expression changes with the appearance of dead or dying cells by several histological procedures. At 12 h, in situ hybridization followed by emulsion dipping showed nonuniform decreases in GluR2 mRNA hybridization grains overlying morphologically healthy-appearing CA3 neurons. GluR1 and N-methyl-D-aspartate receptor mRNAs were unchanged. At 12–16 h, when little argyrophilia or cells with some features of apoptosis were detected by silver impregnation or electron microscopy, single immunohistochemistry with GluR2 and GluR2/3 subunit-specific antibodies demonstrated a pattern of decreased GluR2 receptor protein within CA3 neurons that appeared to predict a pattern of damage, similar to the mRNA observations. Double immunolabeling showed that GluR2 immunofluorescence was depleted and that GluR1 immunofluorescence was sustained in clusters of the same CA3 neurons. Quantitation of Western blots showed increased GluR1:GluR2 ratios in CA3 but not in CA1 or dentate gyrus subfields. Findings indicate that the GluR1:GluR2 protein ratio is increased in a population of CA3 neurons prior to significant cell loss. Data are consistent with the “GluR2 hypothesis” that reduced expression of GluR2 subunits will increase formation of AMPA receptors permeable to Ca²⁺ and predict vulnerability to a particular subset of pyramidal neurons following status epilepticus. Hippocampus 1998;8:511–525. © 1998 Wiley-Liss, Inc.     

Indefatigable CA1 sector neuroprotection with mild hypothermia induced 6 hours after severe forebrain ischemia in rats.

Considerable controversy exists about whether postischemic hypothermia can permanently salvage hippocampal CA1 neurons or just postpone injury. Studies of very brief cooling in rat have found transient benefit, whereas experiments in gerbil using protracted hypothermia report lasting protection. This discrepancy might be because of the greater efficacy of longer cooling or it might, for example, represent an important species difference. In the present study, a 48-hour period of mild hypothermia was induced starting 6 hours after 10 minutes of severe four-vessel occlusion ischemia in rats. Untreated normothermic ischemia resulted in total CA1 cell loss (99%), whereas delayed hypothermia treatment reduced neuronal loss to 14% at a 28-day survival. In unregulated rats, brain temperature spontaneously fell during ischemia, and stayed subnormal for an extended period after ischemia. This mild cooling resulted in more variable and less severe CA1 injury (75%). Finally, vertebral artery cauterization under halothane anesthesia caused an approximately 2 degrees C drop in brain temperature for 1 hour, but prevention of this hypothermia did not significantly affect CA1 damage. In summary, protracted postischemic hypothermia provided robust and long-term CA1 protection in rat. These results encourage the clinical assessment of prolonged hypothermia and its use as a model to understand ischemic CA1 injury.     

Postischemic inhibition of GABA reuptake by tiagabine slows neuronal death in the gerbil hippocampus

The neuroprotective effects of enhancing neuronal inhibition with a γ-aminobutyric acid (GABA) uptake inhibitor were studied in gerbil hippocampus following transient ischemia. We used in vivo microdialysis to determine a suitable dosing regimen for tiagabine (NNC 328) to elevate extracellular levels of GABA within the hippocampus. In anesthetized (normothermic) gerbils, tiagabine (45 mg/kg, i. p.) selectively elevated extracellular GABA levels 450% in area CA1 of the hippocampus. In gerbils subjected to cerebral ischemia via 5-min bilateral carotid occlusion, extracellular GABA levels increased 13-fold in area CA1, returning to baseline within 30–45 min. When tiagabine was injected 10 min following onset of reperfusion, GABA levels remained elevated (200–470%) for 90 min. In addition, tiagabine significantly reduced the ischemic-induced elevation of glutamate levels in area CA1 during the postischemic period when GABA levels were elevated. There was no effect of postischemic tiagabine on aspartate or six other amino acids. Using the same dosing regimen, we evaluated the degree of neuroprotection in the hippocampus of gerbils 4 and 21 days after ischemia. Tiagabine decreased body temperature a maximum of 2.7°C beginning 30 min into reperfusion and lasting 90 min. In untreated gerbils sacrificed 4 and 21 days after ischemia, there was severe necrosis (99%) of the pyramidal cell layer in area CA1. Whereas tiagabine significantly protected the CA1 pyramidal cell layer in ischemic gerbils at 4 days (overt necrosis confined to about 17% of area CA1), the protection diminished significantly 21 days postischemia. When normothermia was maintained both during and after ischemia in a separate group of tiagabine-treated animals, approximately 77% of the CA1 pyramidal cell layer was necrotic at 4 days. Based on these findings, we suggest that (1) tiagabine slows the development of hippocampal degeneration following ischemia, and (2) that mild, postischemic hypothermia is responsible, in large part, for the neuroprotective actions of this drug. We conclude that the histological outcome after administration of cerebral neuroprotectants should be assessed following long-term survival. © 1995 Wiley-Liss, Inc.     

Effects of single or repeated restraint stress on GluR1 and GluR2 flip and flop mRNA expression in the hippocampal formation

The mRNAs encoding the flip and flop isoforms of the glutamate receptor subunits GluR1 and 2 were detected and quantified by in situ hybridization in the hippocampal formation of rats following acute (6h) or chronic (6h daily for 21 days) restraint stress. The GluR1 flip mRNA was slightly reduced in CA1 after chronic stress and the GluR2 flip mRNA was increased in the dentate gyrus (DG), CA4, and CA3 after acute stress. There were no changes in the mRNA encoding the flop isoforms of either GluR1 or 2 in the hippocampus. In entorhinal cortex, the GluR1 flip mRNA was significantly increased after both acute and chronic stress, while the flop isoform increased only after chronic stress. The GluR2 flip mRNA was slightly increased after acute and chronic stress. However, no changes were found for the flop isoform of GluR2. These results suggest that different assembly of AMPA receptors subunits and isoforms may underlie, in a different way, the neuronal plastic changes induced by specific type and intensity of stressful stimuli.