Activation of ATP-sensitive potassium channels prevents the cleavage of cytosolic mu-calpain and abrogates the elevation of nuclear c-Fos and c-Jun expressions after hypoxic-ischemia in neonatal rat brain

The purpose of this study was to determine whether activation of ATP-sensitive K+ (KATP) channels with diazoxide (DIZ) is able to prevent the cleavage of cytosolic mu-calpain and abrogate the elevation of nuclear c-Fos and c-Jun protein (c-Fos, c-Jun) expressions after hypoxic-ischemia (HI) in brain. The model of hypoxic-ischemic brain injury (HIBI) was made in the 7-day-old Sprague-Dawley (SD) rats by left carotid arterial ligation and hypoxia (8% oxygen). DIZ was injected into the left lateral ventricle (5 microl, 1 mg/ml) before or post-hypoxic-ischemia (HI) insults. Western blot and computer image processing were used to detect the integrated density of nuclear c-Fos and c-Jun at 4 h and cleavage of cytosolic mu-calpain at 24 h after HI insults from cerebral cortical and hippocampal samples. Compared with HI controls (c-Fos=30.37+/-7.39 from cortical samples, 58.61+/-3.64 from hippocampal samples; c-Jun=52.48+/-14.23 from cortical samples, 35.55+/-4.73 from hippocampal samples), there was a significant down-regulation of c-Fos and c-Jun expressions from cortical and hippocampal samples in rats treated with DIZ before (c-Fos=11.10+/-4.64 from cortical samples, 4.82+/-3.38 from hippocampal samples; c-Jun=19.01+/-5.29 from cortical samples, 35.55+/-4.73 from hippocampal samples) or post- (c-Fos=18.81+/-7.93 from cortical samples, 11.33+/-7.05 from hippocampal samples; c-Jun=24.64+/-10.01 from cortical samples, 19.75+/-3.47 from hippocampal samples) HI insults. Furthermore, the ratio of 76 kD/80 kD of mu-calpain was down-regulated from cortical and hippocampal samples in rats treated with DIZ before or post-HI insults, demonstrating a significant difference compared with that observed in HI controls. Finally, the increase in DNA fragments caused by the HI injury was decreased or eliminated by the treatment with DIZ. These data suggests that activation of KATP channels by DIZ reduces the degree of mu-calpain proteolysis, and c-Fos and c-Jun expressions in immature brain may contribute to the neuroprotection of K(ATP) channel openers against HIBI.     

Indomethacin ameliorates ischemic neuronal damage in the gerbil hippocampal CA1 sector

The purpose of our experiment was to examine whether the cyclooxygenase inhibitor indomethacin ameliorates neuronal injury in the gerbil hippocampal CA1 sector following 5 minutes of forebrain ischemia. Thirty minutes before bilateral carotid artery occlusion, Mongolian gerbils were injected intraperitoneally with 1 (n = 10), 2 (n = 10), 5 (n = 12), or 10 (n = 7) mg/kg of indomethacin. Seven days after occlusion, the gerbils were perfusion-fixed and neuronal density in the hippocampal CA1 sector was assessed. The mean +/- SEM neuronal density in nine unoperated normal gerbils was 307 +/- 9/mm, in 10 untreated ischemic gerbils 55 +/- 21/mm, and in seven vehicle-treated ischemic gerbils 15 +/- 9/mm. The mean +/- SEM neuronal density in ischemic gerbils treated with 1, 2, 5, or 10 mg/kg indomethacin was 132 +/- 28/mm, 154 +/- 29/mm, 176 +/- 30/mm, and 136 +/- 39/mm, respectively. Indomethacin at any dose significantly ameliorated ischemic neuronal damage in the gerbil hippocampal CA1 sector.     

Angiotensinogen is secreted by pure rat neuronal cell cultures

Previous studies are divided between those which support a neuroglial (astrocyte) source for brain angiotensinogen and those which indicate that both astrocytes and neurones synthesize the precursor of angiotensin II. In this study, separate cultures of astrocytes and neuronal cells were prepared and established as being essentially pure by appropriate immunocytochemical cell markers. Angiotensinogen production by these cultures, as measured by a direct radioimmunoassay, was 20.74 +/- 3.62 ng angiotensinogen/10(6) cells/24 h (mean +/- S.D., n = 8) for astrocytes and 4.39 +/- 0.94 ng/10(6) cells/24 h (mean +/- S.D., n = 29) for neurones. Angiotensinogen secretion from both cell types was unaffected by treatments which stimulate the regulatory secretory pathway by modulating intracellular cAMP levels. In contrast, it was reduced from 23.20 +/- 2.14 to 8.14 +/- 1.31 ng/10(6) cells/24 h (S.E.M., n = 7) in astrocyte cultures by the constitutive pathway inhibitor, monensin. Angiotensinogen secreted by astrocytes and neurones was compared to pure angiotensinogen and that in plasma and cerebrospinal fluid (CSF) by cation-exchange mono S column chromatography. Pure angiotensinogen eluted as two separate peaks corresponding to the major forms of plasma angiotensinogen, whereas angiotensinogen in CSF and culture media coeluted with a third minor form of plasma angiotensinogen. It was concluded that neuronal cells as well as astrocytes secrete angiotensinogen which is distinctly different from plasma angiotensinogen.     

Hyperglycemic exacerbation of neuronal damage following forebrain ischemia: microglial, astrocytic and endothelial alterations

We undertook a detailed characterization of the cellular responses to acute global cerebral ischemia complicated by hyperglycemia. Anesthetized, physiologically monitored male Wistar rats received 12.5 min of global forebrain ischemia by bilateral common carotid artery occlusions plus hemorrhagic hypotension to 45 mm Hg. Cranial temperature was maintained at normothermic levels. Hyperglycemic animals received dextrose (2.5 ml of a 25% solution, intraperitoneally) prior to ischemia; this doubled the mean plasma glucose concentration to 296 mg/100 ml. At 3 days (n = 10) or 24 h (n = 4) after ischemia, brains were perfusion-fixed and paraffin-embedded for light microscopic histopathology and for the histochemical visualization of activated microglia and the immunocytochemical visualization of glial fibrillary acid protein. Normal-neuron counts in the vulnerable hippocampal CA1 sector of hyperglycemic-ischemic (HI) rats were reduced to one-third the number observed in normoglycemic-ischemic (NI) animals. Ischemic cell counts in the striatum were increased fivefold or more in HI compared to NI rats, and normal small-neuron counts were reduced by two-thirds. The neocortex and striatum of NI rats showed only mild damage, while the majority of HI rats had extensive lesions, and several showed large cortical, striatal or thalamic infarcts. In addition, widespread cortical ischemic neuronal changes were evident in HI animals. No endothelial alterations were present in NI rats. By contrast, HI rats showed prominent peri- and intravascular polymorphonuclear and monocytic accumulation evident at 24 h; frequent white cell thrombi in pial arterioles on day 3; and thickening of vascular endothelium, with foci of parenchymal rarefaction or microinfarction adjacent to occluded vessels. Prominent microglial activation, often along the course of penetrating blood vessels, was common in the striatum and neocortex of HI animals but was much less extensive in the NI group. Activated microglia in HI rats were typically hypertrophic and amoeboid. These results suggest that the detrimental influence of hyperglycemia in ischemia is initially mediated by an action on vascular endothelium, which in turn leads to widespread foci of infarction and neuronal loss. Received: 18 March 1998 / Revised, accepted: 2 June 1998     

Differential vulnerability of immature murine neurons to oxygen-glucose deprivation

In vivo studies support selective neuronal vulnerability to hypoxia-ischemia (HI) in the developing brain. Since differences in intrinsic properties of neurons might be responsible, pure cultures containing immature neurons (6-8 days in vitro) isolated from mouse cortex and hippocampus, regions chosen for their marked vulnerability to oxidative stress, were studied under in vitro ischemic conditions-oxygen-glucose deprivation (OGD). Twenty-four hours of reoxygenation after 2.5 h of OGD induced significantly greater cell death in hippocampal than in cortical neurons (67.8% vs. 33.4%, P = 0.0068). The expression of neuronal nitric oxide synthase (nNOS) protein, production of nitric oxide (NO), and reactive oxygen species (ROS), as well as glutathione peroxidase (GPx) activity and intracellular levels of reduced glutathione (GSH), were measured as indicators of oxidative stress. Hippocampal neurons had markedly higher nNOS expression than cortical neurons by 24 h of reoxygenation, which coincided with an increase in NO production, and significantly greater ROS accumulation. GPx activity declined significantly in hippocampal but not in cortical neurons at 4 and 24 h after OGD. The decrease in GSH level in hippocampal neurons correlated with the decline of GPx activity. Our data suggest that developing hippocampal neurons are more sensitive to OGD than cortical neurons. This finding supports our in vivo studies showing that mouse hippocampus is more vulnerable than cortex after neonatal HI. An imbalance between excess prooxidant production (increased nNOS expression, and NO and ROS production) and insufficient antioxidant defenses created by reduced GPx activity and GSH levels may, in part, explain the higher susceptibility to OGD of immature hippocampal neurons.     

Hypoxia-ischemia upregulates TRAIL and TRAIL receptors in the immature rat brain

The immature brain is susceptible to inflammatory injury induced by hypoxia-ischemia (HI) or infection, which causes serious neurodevelopmental disabilities in the survivors of preterm births. Recently, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors (death receptor DR4/5 and decoy receptor DcR1/2) were reported to mediate various neuroinflammatory responses. However, little information is available regarding the role of TRAIL and its receptors in the immature brain after HI. The purpose of this study was to evaluate the expression of TRAIL and its receptors in the immature brain after HI and relate this expression to neurological function. We performed right common carotid artery ligation followed by hypoxia (6% O(2), 37°C) for 2.5 h to induce HI in postnatal day 3 rats. The distribution of TRAIL and its receptors, caspase-3 and CD68-labeled microglia/macrophages was evaluated 24 h after HI by immunostaining. The protein and mRNA expression of TRAIL and DR5 was measured by Western blot and real-time PCR, respectively. Delayed neuronal loss was evaluated by NeuN and Nissl staining 7 days after HI. Furthermore, neurological deficits were evaluated by a righting reflex test, time of eye opening and T-maze test. The expression of TRAIL, DR5 and DcR1/2 receptors and caspase-3 was more pronounced in the ipsilateral hemisphere compared with the contralateral part and the control group 24 h after HI. DR5/active caspase-3 double-positive cells were observed at 24 h after HI in the ipsilateral hemisphere but not in the contralateral hemisphere. The TRAIL and CD68 double-labeled cells were more pronounced in the ipsilateral cortical regions compared with the corresponding regions of the contralateral part. HI also resulted in a significant increase in TRAIL and DR5 protein and mRNA expression at 24 h, which corresponded to neuronal cell loss 7 days after HI. Furthermore, the HI group displayed impaired neurobehavioral development compared with the control group (p < 0.05). Altogether our results show that the TNF-α superfamily ligand TRAIL is induced on CD68+ microglia/macrophages after perinatal HI and that one of its receptors, DR5, is induced on neocortical neurons and glial cells. That many DR5+ cells were also caspase-3+ strongly supports the conclusion that these signaling molecules are involved in the delayed loss of neurons in the neocortex and in the neurobehavioral deficits that are often seen after perinatal HI.     

Temporal profile of expression and cellular localization of inducible nitric oxide synthase, interleukin-1beta and interleukin converting enzyme after cryogenic lesion of the rat parietal cortex.

We used in situ hybridization, RT PCR and immunohistochemistry to study the time course of expression and the cellular localization of inducible nitric oxide synthase (iNOS) and interleukin-1beta (IL-1beta) during the first 7 days after induction of a standardized cryogenic lesion on the right parietal cortex in male rats. Cryogenic lesion induced iNOS mRNA in the lesioned hemisphere after 6 to 72 h with a maximum (15+/-2 cells/mm2, n=4, p<0.01 vs. sham) at 24 h. Microglia, invading monocytes and granulocytes in and around the lesion expressed iNOS immunoreactivity starting at 12 h and peaking (29+/-10 cells/mm2, n=4, p<0.05 vs. sham) at 24 h after lesion. Induction of IL-1beta mRNA expression was immediate with a peak (9+/-1 cells/mm2, n=4, p<0.01 vs. sham) at 24 h after cryogenic lesion. The number of round cells with IL-1beta immunoreactivity around the lesion was maximal (8+/-2 cells/0.1 mm2, n=3, p<0.01 vs. sham) at 24 h. A weak astrocytic expression of IL-1beta-immunoreactivity was seen in sham animal brains. Astrocytic IL-1beta-expression was significantly increased in the lesion hemisphere and both hippocampi. Interleukin converting enzyme (ICE) was expressed in astrocytes and microglia around the lesion 6 h after injury. The number of ICE immunoreactive cells (8+/-2 cells/0. 1 mm2, n=3, p<0.05 vs. sham) peaked at 72 h after lesion. Neuronal expression of ICE and IL-1beta was seen in the lesion periphery 72 h and 7 days after injury. At this time, morphological features of apoptosis were evident in cells in the lesion periphery. The data indicate an early activation of microglia and monocyte invasion into the lesion hemisphere leading to multicellular expression of iNOS, ICE, and IL-1beta. These events may contribute to the expansion of neuronal damage after brain injury.     

Partial hippocampal kindling increases GABAB receptor-mediated postsynaptic currents in CA1 pyramidal cells

In previous studies, we showed that partial hippocampal kindling decreased the efficacy of the presynaptic GABAB receptors on both GABAergic and glutamatergic terminals of CA1 neurons in hippocampal slices in vitro. In this study, GABAB receptor-mediated inhibitory postsynaptic currents (GABAB-IPSCs) were assessed by whole-cell recordings in CA1 pyramidal neurons in hippocampal slices of male Long-Evans rats. The peak GABAB-IPSC evoked by a brief train of supramaximal stratum radiatum stimuli (20 pulses of 300 Hz) in the presence of picrotoxin (0.1 mM) and kynurenic acid (1 mM) was larger in neurons of kindled (65.9 +/- 5.2 pA, N=42 cells) than control (45.8 +/- 4.8 pA, N=32 cells) rats (P<0.01). Adding GABA uptake blocker nipecotic acid (1 mM) or GABAB receptor agonist baclofen (0.01 mM) in the perfusate induced outward currents that were blocked by GABAB receptor antagonist CGP 55845A (1 microM). The peak outward current induced by nipecotic acid was larger in neurons of the kindled (55.4 +/- 5.7 pA, N=30) than the control group (39.8 +/- 4.5 pA, N=28) (P<0.05). However, the magnitude of the baclofen-induced current was not different between kindled (90.8 +/- 6.9 pA, N=29) and control (87.2 +/- 5.9 pA, N=21) groups (P>0.05). We concluded that partial hippocampal kindling increased GABAB-IPSCs in hippocampal CA1 pyramidal cells via multiple presynaptic mechanisms.     

A phospholipase C inhibitor ameliorates postischemic neuronal damage in rats.

BACKGROUND AND PURPOSE: The hypothesis of calcium-induced neuronal damage has been proposed regarding brain ischemia. Phospholipase C is an enzyme that catalyzes the phosphodiesteratic cleavage of phosphatidylinositol. The cleavage of phosphatidylinositol 4,5-bisphosphate by phospholipase C yields 1,4,5-inositol triphosphate, which mediates intracellular release of calcium, and 1,2-diacylglycerol, which is an activator of protein kinase C. We examined the effect of phenylmethylsulfonyl fluoride, a phospholipase C inhibitor, on delayed neuronal damage after transient forebrain ischemia in the hippocampal CA1 subfield in rats to assess the role of phospholipase C in postischemic neuronal damage. METHODS: Twenty-minute forebrain ischemia was induced using the method of Pulsinelli and Brierley. We measured the neuronal density of the hippocampal CA1 subfield 7 days after reperfusion. The effect of phenylmethylsulfonyl fluoride was tested in both pretreatment and posttreatment groups. RESULTS: In the vehicle treatment group (n = 13), neuronal density was 51 +/- 42/mm (mean +/- SD). The neuronal densities in the 50-mg/kg (n = 12) and 100-mg/kg (n = 14) phenylmethylsulfonyl fluoride pretreatment groups and the 100-mg/kg (n = 10) phenylmethylsulfonyl fluoride posttreatment group were 99 +/- 50, 150 +/- 55, and 143 +/- 63/mm, respectively. These values were significantly higher than that of the vehicle treatment group (p less than 0.05, p less than 0.01, and p less than 0.01, respectively). CONCLUSIONS: It is suggested that the activation of phospholipase C has an important role in postischemic delayed neuronal damage.     

Hippocampal progenitor cells express nestin following cerebral ischemia in rats

This study addresses whether hippocampal progenitor cells express nestin following cerebral ischemia in rats. Cell counts within the hippocampal hilus were significantly greater following severe (eight-vessel occlusion) ischemia than following intermediate (four-vessel occlusion) ischemia (1527+/-87/mm2 vs. 918+/-71/mm2). Bromedeoxyuridine-positive cell counts were significantly higher with severe ischemia than with intermediate ischemia or in sham-operated animals, respectively (368+/-45, 43+/-14 and 7+/-1/mm2). In the eight-vessel occlusion group, 47+/-8/mm2 bromedeoxyuridine-labeled cells expressed nestin, significantly higher than in the four-vessel occlusion group and sham-operated animals (1+/-1 and 1+/-0/mm2, P<0.01 vs. eight-vessel occlusion, respectively). Confocal microscopy verified that a subset of the bromedeoxyuridine-positive cells expressed nestin. In conclusion, severe ischemia elicits nestin expression in hippocampal progenitor cells in rats.     

Lovastatin reduces neuronal cell death in hippocampal CA1 subfield after pilocarpine-induced status epilepticus: preliminary results

OBJECTIVE: To further characterize the capacity of lovastatin to prevent hippocampal neuronal loss after pilocarpine-induced status epilepticus (SE) METHOD: Adult male Wistar rats were divided into four groups: (A) control rats, received neither pilocarpine nor lovastatin (n=5); (B) control rats, received just lovastatin (n=5); (C) rats that received just pilocarpine (n=5); (D) rats that received pilocarpine and lovastatin (n=5). After pilocarpine injection (350 mg/kg, i.p.), only rats that displayed continuous, convulsive seizure activity were included in our study. Seizure activity was monitored behaviorally and terminated with an injection of diazepam (10 mg/kg, i.p.) after 4 h of convulsive SE. The rats treated with lovastatin received two doses of 20 mg/kg via an oesophagic probe immediately and 24 hours after SE induction. Seven days after pilocarpine-induced SE, all the animals were perfused and their brains were processed for histological analysis through Nissl method. RESULTS: The cell counts in the Nissl-stained sections performed within the hippocampal formation showed a significant cell loss in rats that received pilocarpine and presented SE (CA1=26.8 +/- 13.67; CA3=38.1 +/- 7.2; hilus=43.8 +/- 3.95) when compared with control group animals (Group A: CA1=53.2 +/- 9.63; CA3=63.5 +/- 13.35; hilus=59.08 +/- 10.24; Group B: CA1=74.3 +/- 8.16; CA3=70.1 +/- 3.83; hilus=70.6 +/- 5.10). The average neuronal cell number of CA1 subfield of rats that present SE and received lovastatin (44.4 +/- 17.88) was statically significant increased when compared with animals that just presented SE. CONCLUSION: Lovastatin exert a neuroprotective role in the attenuation of brain damage after SE.     

Vasopressin and oxytocin in brain areas of rats selectively bred for differences in behavioral performance.

Wistar rats were selectively bred over 10 generations for differences in performance in a footshock-motivated brightness discrimination (BD) test in a Y-maze. High behavioral performance (Wis/HBP) and low behavioral performance (Wis/LBP) rat lines were obtained which differ significantly in all behavioral components tested: frequency of correct responses, number of trials to criterion, response latency (HBP less than LBP), and frequency of freezing behavior (HBP less than LBP), the latter suggesting differences in emotionality. In Wis/LBP rats, furthermore, the normal increase in behavioral performance between the training and the relearning session, which indicates the formation of a memory trace, disappeared during selection. In male breeders sampled during selection of the two lines (Wis/HBP: n = 17; Wis/LBP: n = 21), both arginine vasopressin (AVP) and oxytocin (OXT) contents were measured by radioimmunoassay in the motor cortex, septum/striatum, hippocampus, hypothalamus, medulla oblongata and posterior pituitary. Compared with the Wis/HBP rats, the Wis/LBP rats contained less AVP in the hippocampus (3.1 +/- 0.58 vs 8.3 +/- 1.4 pg/mg wet wt., mean +/- S.E.M., P less than 0.001), but more AVP in the medulla (1.7 +/- 0.20 vs 1.1 +/- 0.18 pg/mg, P less than 0.05). In contrast, no significant differences between the lines were detected with respect to OXT concentrations. In the Wis/LBP rats, moreover, the hippocampal AVP content decreased during selection (r = -0.645, P less than 0.01), while the acquisition response latency increased (r = 0.549, P less than 0.01). As a consequence, a significant, albeit weak, negative correlation (r = -0.483, P less than 0.05) was observed between the individual hippocampal AVP content and the response latency during acquisition. Thus, the results confirm the view that genetically determined differences in the hippocampal content of endogenous AVP may contribute to an individual's level of emotionality and behavioral performance.     

Increased severity of acute cerebral ischemic injury correlates with enhanced stem cell induction as well as with predictive behavioral profiling

An 8-vessel-occlusion (8VO) method was developed to compare with the conventional 4-vessel-occlusion (4VO) in hippocampal ischemic damage and progenitor cell induction 10 days following ischemia in female rats. Eight posture-relevant tests were performed following ischemia to correlate grades of postural abnormality with the histological outcome. The total hippocampal living cell ratio including 7 hippocampal subregions in 8VO group (n=11) was much lower than that in 4VO group (n=10, 51+/-5% vs. 78+/-4, p<0.01). In 4VO group, BrdU positive cells were mainly located in the subgranular zone (SGZ) with a count of 54+/-20/mm2 (7micro-thick slice), comparable to the maximal level following global ischemia in male gerbils and rats reported so far referring to slice-thickness differences (50-60 micro-thick slices). Similarly, nestin-bearing cells were 29+/-11/ mm2. In 8VO group, BrdU and nestin positive cells increased by 10 times. Triple staining of BrdU, nestin and DAPI demonstrated that BrdU-immunoreactivity was extensively distributed in the hippocampal hilus while the nestin was mainly located along the SGZ. Most of nestin labeling was not co-localized with the BrdU, indicating that establishment of these cells might precede BrdU injections (8 and 9d post ischemia). Behavioral scores were much greater for 8VO group than for 4VO group and composite postural scores well correlated with the hippocampal cell loss. In conclusion, severe ischemia correlates with vigorous induction of the hippocampal progenitor cells in rats while behavioral profiling of posture changes permits prediction of severity of damage.     

Dicyclomine, an M1 muscarinic antagonist, reduces infarct volume in a rat subdural hematoma model

The rat subdural hematoma (SDH) model produces a zone of ischemic brain damage within the hemisphere beneath the SDH. Previous studies have measured large increases in extracellular acetylcholine during cerebral ischemia in the rat. We examined infarct volume after selectively blocking muscarinic M1 receptors with dicyclomine during SDH. Rats were anesthetized with isoflurane (2%), intubated, and femoral artery and vein cannulated. Autologous blood (0.375 ml) was injected (0.05 ml/min) under the dura of the right parietal cortex. Dicyclomine (5 mg/kg, i.v.) was injected at 5 min after and again at 2 h after completion of the subdural blood infusion. Blood pressure and intracranial pressure (ICP) were continuously measured. At 4 h after SDH rats were euthanized, brains sectioned, and immunoreacted with glia fibrillary acidic protein. Cortical infarct volume was quantified in coronal brain sections at 0.7-mm intervals from +1.0 mm to -3.9 mm relative to bregma. Infarct volume in drug-treated rats (n = 10) 22.1 +/- 6.99 mm3 was significantly smaller (p < 0.02) than vehicle treated rats (n = 10) 56.7 +/- 9.59 mm3. ICP, blood pressure and cerebral perfusion pressure were not significantly different between groups. These data suggest that activation of M1 muscarinic receptors during an ischemic event may contribute to the development of subsequent pathology.     

Persistent neuroprotection with prolonged postischemic hypothermia in adult rats subjected to transient middle cerebral artery occlusion

Postischemic hypothermia provides long-lasting neuroprotection against global cerebral ischemia in adult rats and gerbils. Studies indicate that hypothermia must be prolonged (e.g., 24 h) to indefatigably salvage hippocampal CA1 neurons. Delayed hypothermia also reduces focal ischemic injury. However, no study has examined long-term outcome following postischemic hypothermia in adult animals. Furthermore, most studies examined only brief hypothermia (e.g., 3 h). Since previous studies may have overestimated long-term benefit and have likely used suboptimal durations of hypothermia, we examined whether prolonged cooling would attenuate infarction at a 2-month survival time following middle cerebral artery occlusion (MCAo) in rats. Adult male Wistar rats were implanted with telemetry brain temperature probes and later subjected to 30 min of normothermic MCAo (contralateral to side of probe placement) or sham operation. Ischemia was produced by the insertion of an intraluminal suture combined with systemic hypotension (60 mm Hg). Sham rats and one ischemic group controlled their own postischemic temperature while another ischemic group was cooled to 34 degrees C for 48 h starting at 30 min following the onset of reperfusion. The infarct area was quantified after a 2-month survival time. Normothermic MCAo resulted in almost complete striatal destruction (91% loss +/- 12 SD) with extensive cortical damage (36% +/- 16 SD). Delayed hypothermia treatment significantly reduced cortical injury to 10% +/- 10 SD (P < 0.001) while striatal injury was marginally reduced to 79% loss +/- 17 SD (P < 0.05). Delayed hypothermia of only 34 degrees C provided long-lasting cortical and striatal protection in adult rats subjected to a severe MCAo insult. These results strongly support the clinical assessment of hypothermia in acute stroke.     

Ovarian steroid modulation of seizure severity and hippocampal cell death after kainic acid treatment

To determine whether maintained estrogen or progesterone levels affect kainic acid (KA) seizure patterns or the susceptibility of hippocampal neurons to death from seizures, ovariectomized Sprague-Dawley rats were implanted with estrogen pellets, 0.1 or 0.5 mg, that generated serum levels of 42.4 +/- 6.6 (mean +/- SEM) and 242.4 +/- 32.6 pg/ml or one to six capsules of progesterone that generated serum levels of 11.00 +/-.72 to 48.62 +/- 9.4 ng/ml. Seven days later, the rats were administered KA (8.5mg/kg, ip) and scored for seizure activity; 96 h later, the rats were killed and their brains processed for localization of neuron nuclear antigen (NeuN), a general neuronal marker. The hippocampus was scored for spread (the number of separate regions showing cell loss), and the area within the CA fields occupied by NeuN immunoreactivity was measured (indicating surviving neurons). Administration of estrogen or progesterone (independent of dose) significantly reduced mortality from KA seizures. Progesterone reduced seizure severity in animals that received one to four implants; compared with controls, no difference in seizure severity was noted for animals with six progesterone implants. The reduced seizures in progesterone-treated animals were accompanied by a reduction in the spread of hippocampal damage (r(2) = 0.87; P < 0.05). Likewise, in progesterone-treated rats, neuron survival and reduction in seizure scores were correlated (r(2) = 0.76; P < 0.0001). Estrogen had no effect on seizure severity (P > 0.05), but reduced both the spread (P < 0.05) and degree of neuronal loss (P < 0.05). Indeed, in the estrogen-treated rats, neuronal death was significantly lower than that observed in progesterone-treated animals with equally severe seizures (P < 0.05). These data are consistent with the hypothesis that progesterone produces its effects by reducing seizures, whereas estrogen has little beneficial effect on seizure behavior but protects the hippocampus from the damage seizures produce.     

Differential effects of glucocorticoids and gonadal steroids on glutathione levels in neuronal and glial cell systems

The aim of the present study was to investigate the short- and long-term effects of glucocorticoids [corticosterone (CORT), dexamethasone (DEX), 6-methylprednisolone (6-MP)] and gonadal steroids [17beta-estradiol (E(2)), progesterone (PROG), testosterone (TEST)] on the levels of the antioxidant glutathione (GSH) in different cell systems of the CNS (neuronal hippocampal HT22 cells, primary hippocampal and neocortical brain cells, and C(6) glioma cells). In HT22 cells, steroids exerted mainly long-term effects. Significant increases of GSH levels were detectable after a 24 hr treatment with 10(-7) M of DEX (122% +/- 5%), 6-MP (208% +/- 32%), E(2) (134% +/- 10%), and TEST (155% +/- 17%). A significant decrease occurred after incubation with PROG for 24 hr (79% +/- 9%). In primary hippocampal cultures, a 24 hr treatment with DEX (140% +/- 8%), E(2) (123% +/- 6%), and PROG (118% +/- 5%) led to significant increases of the GSH levels, whereas, in neocortical primary cultures, only an incubation with E(2) increased GSH (149% +/- 8%). In C(6) cells, hormone treatment led to both significant short-term (1 hr: CORT 114% +/- 5%, DEX 90% +/- 3%, E(2) 88% +/- 3%; 3 hr: DEX 115% +/- 5%, E(2) 122% +/- 6%, TEST 78% +/- 4%) and significant long-term (24 hr: CORT 74% +/- 4%, 6-MP 84% +/- 5%, E(2) 115% +/- 6%, PROG 91% +/- 4%, TEST 116% +/- 5%) effects. In summary, we were able to demonstrate differential effects of steroids on GSH levels in different cellular CNS models, showing an important influence of steroids and especially E(2) on antioxidative cellular functions in neuronal and glial cells.     

The effect of MK-801 on cortical spreading depression in the penumbral zone following focal ischaemia in the rat.

Cortical spreading depression (CSD) is a transient depression of neuronal activity that spreads across the cortical surface. In the present studies, we have investigated CSD activity in the penumbral zone following permanent middle cerebral artery (MCA) occlusion in the rat (n = 16/group), using double-barreled Ca(2+)-sensitive microelectrodes. Measurements of CSD activity were made for 3 h in each animal. During this time, a varying number of spontaneous CSDs were seen in the control group (total was 30, with a range of 0-7/rat). These CSDs were of varying duration: "small" (approximately 1 min) and "big" (5-45 min) CSDs. During a CSD, the extracellular [Ca2+] decreased to 0.11 +/- 0.07 mM (mean +/- SD). After 3 h, the extracellular [Ca2+] in the cortex (penumbral zone) was either normal (10/16 rats) or lowered to 0.5 mM (2/16 rats) or to 0.1 mM (4/16 rats). In the caudate nucleus (ischaemic core area), all rats had an extracellular [Ca2+] of approximately 0.1 mM when measured after the 3 h recording period. Neuropathological evaluation of the brains of the animals, which had been allowed to survive for 24 h after MCA occlusion, revealed ischaemic damage in the dorsolateral cortex and caudate nucleus. Administration of the noncompetitive NMDA antagonist, MK-801 (3 mg/kg i.p.), 30 min after MCA occlusion resulted in 24 and 29% reductions in the volume of hemispheric and cortical damage, respectively, which was highly significant (p less than 0.0001); no protection was seen against caudate damage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intrinsic optical imaging reveals regionally different manifestation of spreading depression in hippocampal and entorhinal structures in vitro

The spatiotemporal features of spreading depression (SD) were analyzed in vitro by using combined hippocampal-entorhinal cortex slices. SDs were induced by microinjection of 1 M KCl in the stratum radiatum of the CA1 region of the hippocampus. Measurements of extracellular field potentials, extracellular space (ECS) volume changes and intrinsic optical signal changes were combined to study SD features in different regions of the slice. Each SD was associated with a pronounced shrinkage of the extracellular space (ECS) volume and a decrease in light transmittance. The beginning of the optical signal change occurred simultaneously with the electrographic onset as measured with extracellular microelectrodes but outlasted the dc shift for tens of seconds. The amplitude of the intrinsic optical signal change displayed marked regional variations with greatest changes of 12% in cortical regions. The signal amplitudes were considerably lower in hippocampal regions. The analysis of spread patterns revealed two types of waves: fully propagated waves spreading from CA1 all the way to the temporal neocortex and abortive waves that ceased earlier. The spread velocities displayed pronounced regional differences with highest velocities of 5.4 +/- 0.3 mm/min in the area CA3 of the hippocampal formation and lowest velocities of 2.7 +/- 0.1 mm/min in cortical regions.     

A correlative study of calcium channel antagonist binding and local neuropathological features in the hippocampus in Alzheimer''s disease

[3H]PN200-110 binding sites were studied by means of quantitative autoradiography in hippocampal sections of patients with Alzheimer's disease and age-matched control subjects. Choline acetyltransferase activity, plaque, tangle and cell densities were also determined in the same tissue samples used for autoradiographic studies. Quantitative autoradiographic analysis of [3H]PN200-110 binding in control hippocampus revealed a heterogeneous pattern similar to that described in rodents, being particularly high in the dentate gyrus. In Alzheimer's disease, [3H]PN200-110 binding was markedly reduced in the subiculum (control = 9.85 +/- 1.41 pmol/g; Alzheimer = 3.41 +/- 0.54 pmol/g, mean +/- S.E.M., P less than 0.001). In the subiculum there was a disproportionate reduction of [3H]PN200-110 binding in comparison to cell loss in Alzheimer's disease. The activity of choline acetyltransferase in the hippocampus was markedly reduced in Alzheimer's disease (controls 6.9 +/- 1.0; Alzheimer 2.7 +/- 0.9 nmol/h/mg protein, mean +/- S.E.M., P less than 0.01). There was a strong correlation between choline acetyltransferase activity and [3H]PN200-110 binding in the subiculum. [3H]PN200-110 binding did not correlate with plaque density in the subiculum. The discrete reduction and preservation of [3H]PN200-110 binding in the present study is consistent with the pattern of selective cellular vulnerability in the hippocampal region in Alzheimer's disease.