Post-ischemic administration of nimodipine following focal cerebral ischemic-reperfusion injury in rats alleviated excitotoxicity, neurobehavioural alterations and partially the bioenergetics

The present study focuses on the temporal calcium significance in middle cerebral artery occluded (2 h ischemia)-reperfused (70 h reperfusion) rats treated with nimodipine (NM) through concurrent measurements of excitotoxicity, bioenergetics and neurobehavioural paradigms. Further, the suitable therapeutic time window of calcium channel antagonism in stroke was also ascertained. NM (5 mg/kg, i.p.) was administered at pre (30 min before the induction of ischemia), during (1 h following occlusion of MCA) and post-ischemic (3 h after begin of reperfusion) states. The magnitude of neuroprotection in terms of excitotoxicity (glutamate, glutamine synthetase, Na⁺K⁺ATPase), bioenergetics (ATP, NAD⁺) and neurobehavioural paradigms (neurological score and open field exploratory behaviour) were measured and compared to ensure the therapeutic time-window of NM in stroke. Middle cerebral artery occlusion-reperfusion (MCAO/R) was found to elevate glutamate, glutamine synthetase levels and deplete Na⁺K⁺ATPase activity in the vehicle treated group (IR group). Significant decrease in bioenergetics such as ATP and NAD⁺ levels was also observed. Further, IR group demonstrated grievous oxidative stress (increase in lipid peroxidation, protein carbonyl content, nitrite/nitrate levels and decrease in superoxide dismutase and glutathione levels) along with anxiogenic behaviour, neurological deficits and neuronal damage and decreased nuclear to cytoplasm ratio in CA1 hippocampal region. Post-ischemic NM administration reversed the excitotoxicity, neurobehavioural and histopathological alterations significantly, but it restored bioenergetics level in MCAO/R rats only partially.These findings were further confirmed with the combination treatment (CT) of post-ischemic NM and pre-ischemic memantine (MN) administration, since MN showed protective effect in the pre-ischemic administration (Babu and Ramanathan, 2009). The failure of NM to forefend the neurodegeneration on pre- and during-ischemic administration suggests that the initial phase damages in ischemic-reperfusion (IR) might be mediated through other mechanism(s) such as glutamergic overstimulation or reverse operation of glutamate transporters. From the present study, it is concluded that calcium plays a crucial role in post-ischemic status and the suitable therapeutic time window of calcium antagonism is the post-ischemic state.     

Adult responses to an ischemic stroke in a rat model of neonatal stress and morphine treatment

Critically ill newborn infants experience stressors that may alter brain development. Using a rodent model, we previously showed that neonatal stress, morphine, and stress plus morphine treatments each influence early gene expression and may impair neurodevelopment and learning behavior. We hypothesized that the combination of neonatal stress with morphine may alter neonatal angiogenesis and/or adult cerebral blood vessel density and thus increase injury after cerebral ischemia in adulthood. To test this, neonatal Lewis rats underwent 8 h/d maternal separation, plus morning/afternoon hypoxia exposure and either saline or morphine treatment (2 mg/kg s.c.) from postnatal day 3–7. A subset received bromodeoxyuridine to track angiogenesis. Adult brains were stained with collagen IV to quantify cerebral blood vessel density. To examine vulnerability to brain injury, postnatal day 80 adult rats underwent right middle cerebral artery occlusion (MCAO) to produce unilateral ischemic lesions. Brains were removed and processed for histology 48 h after injury. Brain injury was assessed by histological evaluation of hematoxylin and eosin, and silver staining. In contrast to our hypothesis, neither neonatal morphine, stress, nor the combination affected cerebral vessel density or MCAO-induced brain injury. Neonatal angiogenesis was not detected in adult rats possibly due to turnover of endothelial cells. Although unrelated to angiogenesis, hippocampal granule cell neurogenesis was detected and there was a trend (P = 0.073) toward increased bromodeoxyuridine incorporation in rats that underwent neonatal stress. These findings are discussed in contrast to other data concerning the effects of morphine on cerebrovascular function, and acute effects of morphine on hippocampal neurogenesis.     

PET imaging of serotoninergic neurotransmission with [11C]DASB and [18F]altanserin after focal cerebral ischemia in rats

The use of selective serotonin reuptake inhibitors has shown functional improvement after stroke. Despite this, the role of serotoninergic neurotransmission after cerebral ischemia evolution and its involvement in functional recovery processes are still largely unknown. For this purpose, we performed in parallel in vivo magnetic resonance imaging and positron emission tomography (PET) with [¹¹C]DASB and [¹⁸F]altanserin at 1, 3, 7, 14, 21, and 28 days after middle cerebral artery occlusion (MCAO) in rats. In the ischemic territory, PET with [¹¹C]DASB and [¹⁸F]altanserin showed a dramatic decline in serotonin transporter (SERT) and 5-HT_2A binding potential in the cortex and striatum after cerebral ischemia. Interestingly, a slight increase in [¹¹C]DASB binding was observed from days 7 to 21 followed by the uppermost binding at day 28 in the ipsilateral midbrain. In contrast, no changes were observed in the contralateral hemisphere by using both radiotracers. Likewise, both functional and behavior testing showed major impaired outcome at day 1 after ischemia onset followed by a recovery of the sensorimotor function and dexterity from day 21 to day 28 after cerebral ischemia. Taken together, these results might evidence that SERT changes in the midbrain could have a key role in the functional recovery process after cerebral ischemia.     

Pre-treatment of adrenomedullin suppresses cerebral edema caused by transient focal cerebral ischemia in rats detected by magnetic resonance imaging

Recent studies suggest the protective effects of adrenomedullin (AM) on ischemic brain damage. The present study was aimed at investigating the effects of AM and its receptor antagonist, AM_22-52, on ischemia-induced cerebral edema and brain swelling in rats using magnetic resonance imaging. Rats were subjected to 60 min of middle cerebral artery occlusion (MCAO) followed by reperfusion. Intravenous injection of AM (1.0 μg/kg), AM_22-52 (1.0 μg/kg), or saline was made before MCAO. Effects of AM injection just after reperfusion were also investigated. One day after ischemia, increases in T₂-weighted signals in the brain were clearly observed. Total edema volume, as well as brain swelling, was greatly and significantly reduced by pre-treatment of AM (reduced by 53%). Extent of brain swelling was significantly correlated with the volume of cerebral edema. The protective effect of AM against edema was more clearly observed in the cerebral cortex (reduced by 63%) than the striatum (reduced by 31%). Increased T₂ relaxation time in the cortex was recovered partially by pre-treatment of AM. Post-treatment of AM had no effects. Pre-treatment of AM_22-52 tended to exacerbate the edema. In another line of experiment, cocktail administration of AM with melatonin, a pineal product having neuroprotective potential as a free radical scavenger, failed to enhance the protective effects of AM alone. The present study clearly suggests the prophylactic effects of AM against cerebral edema, especially the cortical edema, in a rat stroke model.     

Flavonoids from Scutellaria baicalensis Georgi are effective to treat cerebral ischemia/reperfusion

Based on previous studies that have shown flavonoids from the stems and leaves of Scutellaria baicalensis Georgi are neuroprotective agents in a naturally senile, D-galactose, aging in vivo model, as well as an in vitro model of oxidative/hypoxic injury, we established a cerebral ischemia/reperfusion model in rats by middle cerebral artery occlusion. The light/electron microscopic observations found significant neuropathological changes including neuron loss or swelling and rough endoplasmic reticulum injury. Moreover, the activities of lactate dehydrogenase, Na⁺-K⁺-ATPase, Ca²⁺-ATPase and superoxide dismutase were significantly lowered, and the levels of malonaldehyde increased. In addition, the memory of rats worsened. However, treatment with flavonoids from Scutellaria baicalensis Georgi (35, 70 and 140 mg/kg) for 13 days dramatically improved the above abnormal changes. These results suggest that the ability of flavonoids from Scutellaria baicalensis Georgi in attenuating cerebral functional and morphological consequences after cerebral ischemia/reperfusion may be beneficial for the treatment of ischemic brain disease.     

Cerebroprotective action of a Na/Ca channel blocker NS-7: I. Effect on the cerebral infarction and edema at the acute stage of permanent middle cerebral artery occlusion in rats

The effect of a novel Na⁺/Ca²⁺ channel blocker NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy)pyrimidine hydrochloride] on the cerebral infarction, edema and brain energy metabolism was investigated in rats after permanent middle cerebral artery occlusion (MCAO). The infarction and brain water content were evaluated at 48 h and 24 h after MCAO, respectively. A single bolus injection of NS-7 (0.03125–0.25 mg/kg) immediately after MCAO produced a dose-dependent reduction in the infarct volume as well as edema both in the cerebral cortex and striatum. Glycerol (4 g/kg) also decreased water content both in the occluded and non-occluded brain, but it did not reduce the size of cerebral infarction. Unlike glycerol, NS-7 did not change the water content in non-occluded brain. Moreover, a significant protective action was still observed even when NS-7 was injected once at 12 h after occlusion. In addition, NS-7 significantly reversed the decrease in tissue ATP content observed at 3 h but not at 0.5 h after MCAO. These findings suggest that a Na⁺/Ca²⁺ channel blocker NS-7 protects cerebral tissues against ischemic insults by improving the disturbance of cerebral energy metabolism and suppressing the cerebral edema.     

Pyrrolidine dithiocarbamate attenuates brain Aβ increase and improves long-term neurological outcome in rats after transient focal brain ischemia

Evidence suggests an association between brain ischemia and Alzheimer's disease (AD) development. Amyloid plaques consisted of β-amyloid peptide (Aβ) in the brain are a pathological hallmark of AD. Little is known about how brain ischemia induces AD-like neuropathology. A strategy effective to block such brain changes has not been reported. Here, adult male Sprague-Dawley rats were subjected to a 90-min right middle cerebral artery occlusion (MCAO). Pyrrolidine dithiocarbamate (PDTC) at various doses was given daily via gastric gavage with the first dose given at 10 min after the onset of reperfusion. The MCAO increased Aβ1-42 concentrations in the ischemic brain tissues. PDTC attenuated this increase. PDTC also decreased the ischemia-reduced expression of neprilysin, an Aβ degrading enzyme. Aβ1-42 levels were negatively correlated with neprilysin protein abundance. Brain ischemia decreased the expression of β-amyloid converting enzyme 1, a key enzyme to produce Aβ, and increased the expression of insulin-degrading enzyme, another Aβ degrading enzyme. Animals had impaired learning and memory at 2 months after the MCAO. PDTC attenuated this impairment. PDTC also improved long-term neurological outcomes. Our findings suggest that PDTC improves long-term neurological outcome of rats after transient focal brain ischemia. PDTC reduces ischemia-induced Aβ accumulation, possibly via preserving neprilysin expression.     

Morphological and neuro-behavioral parallels in the rat model of stroke

Middle cerebral artery occlusion (MCAO) is widely used as a rat model of focal brain ischemia. Evaluation of brain damage often includes the morphological analysis of the injury area, MRI, and various scales which depend on functional tests, commonly known as neurological severity score (NSS). We determined the optimal number of NSS tests and assessed their capacity for non-invasive evaluation of brain ischemic injury in the rat MCAO model. 275 male Sprague-Dawley rats were randomly divided into five groups, given either permanent (p) MCAO or transient (t) MCAO using an uncoated 4-0 monofilament catheter or a silicone-coated monofilament. The rats’ neurological status was examined before and at 1 and 24 h following MCAO. The size of brain injury was then measured histologically and the extent of right cerebral hemisphere edema was calculated. We established a correlation between these tests and morphological data for brain injury. Adjusted R² of the prediction of total histology score was 0.7. The Hosmer-Lemeshow p-value of this model was 0.812 for total brain histology. For the brain edema the adjusted R² of the prediction model was 0.48. The Hosmer-Lemeshow p-value of this model was 0.558 for brain edema. Our methods of estimating infarct size produces reliable and well correlated results at 24 h and demonstrates to be an easy and quick way to assess infarct size soon after ischemic injury has occurred. The described method for neurological assessment could ultimately aid in assessing various treatment modalities in the early hours following stroke.     

Altered platelet calsequestrin abundance,Na/Ca exchange and Ca signaling responses with the progression of diabetes mellitus

Introduction

Downregulation of calsequestrin (CSQ), a major Ca^2 + storage protein, may contribute significantly to the hyperactivity of internal Ca^2 + ([Ca^2 +]_i) in diabetic platelets. Here, we investigated changes in CSQ-1 abundance, Ca^2 + signaling and aggregation responses to stimulation with the progression of diabetes, especially the mechanism(s) underlying the exaggerated Ca^2 + influx in diabetic platelets.

Materials and methods

Type 1 diabetes was induced by streptozotocin in rats. Platelet [Ca^2 +]_i and aggregation responses upon ADP stimulation were assessed by fluorescence spectrophotometry and aggregometry, respectively. CSQ-1 expression was evaluated using western blotting.

Results

During the 12-week course of diabetes, the abundance of CSQ-1, basal [Ca^2 +]_i and ADP-induced Ca^2 + release were progressively altered in diabetic platelets, while the elevated Ca^2 + influx and platelet aggregation were not correlated with diabetes development. 2-Aminoethoxydiphenyl borate, the store-operated Ca^2 + channel blocker, almost completely abolished ADP-induced Ca^2 + influx in normal and diabetic platelets, whereas nifedipine, an inhibitor of the nicotinic acid adenine dinucleotide phosphate receptor, showed no effect. Additionally, inhibition of Na⁺/Ca^2 + exchange induced much slower Ca^2 + extrusion and more Ca^2 + influx in normal platelets than in diabetic platelets. Furthermore, under the condition of Ca^2 +-ATPase inhibition, ionomycin caused greater Ca^2 + mobilization and Ca^2 + influx in diabetic platelets than in normal platelets.

Conclusions

These data demonstrate that platelet hyperactivity in diabetes is caused by several integrated factors. Besides the downregulation of CSQ-1 that mainly disrupts basal Ca^2 + homeostasis, insufficient Na⁺/Ca^2 + exchange also contributes, at least in part, to the hyperactive Ca^2 + response to stimulation in diabetic platelets.     

l-Dopa effect on frequency-dependent depression of the H-reflex in adult rats with complete spinal cord transection

This study investigated whether l-dopa (DOPA), locomotor-like passive exercise (Ex) using a motorized bicycle exercise trainer (MBET), or their combination in adult rats with complete spinal cord transection (Tx) preserves and restores low frequency-dependent depression (FDD) of the H-reflex. Adult Sprague-Dawley rats (n = 56) transected at T8-9 had one of five treatments beginning 7 days after transection: Tx (transection only), Tx + Ex, Tx + DOPA, Tx + Ex + DOPA, and control (Ctl, no treatment) groups. After 30 days of treatment, FDD of the H-reflex was tested. Stimulation of the tibial nerve at 0.2, 1, 5, and 10 Hz evoked an H-reflex that was recorded from plantar muscles of the hind paw. No significant differences were found at the stimulation rate of 1 Hz. However, at 5 Hz, FDD of the H-reflex in the Tx + Ex, Tx + DOPA and Ctl groups was significantly different from the Tx group (p < 0.01). At 10 Hz, all of the treatment groups were significantly different from the Tx group (p < 0.01). No significant difference was identified between the Ctl and any of the treatment groups. These results suggest that DOPA significantly preserved and restored FDD after transection as effectively as exercise alone or exercise in combination with DOPA. Thus, there was no additive benefit when DOPA was combined with exercise.     

Cerebral oxygen metabolism of rats using injectable 15O-oxygen with a steady-state method

To develop a less-stressful and simple method for measurement of the cerebral metabolic rate of oxygen (CMRO₂) in small animals, the steady-state method was applied to injectable ¹⁵O₂-PET (¹⁵O₂-positron emission tomography) using hemoglobin-containing vesicles (¹⁵O₂-HbV). Ten normal rats and 10 with middle cerebral arterial occlusion (MCAO) were studied using a small animal PET scanner. A series of ¹⁵O-PET scans with C¹⁵O-labeled HbV, H₂¹⁵O, and ¹⁵O₂-HbV were performed with 10 to 15 minutes intervals to measure cerebral blood volume (CBV), cerebral blood flow (CBF), and CMRO₂. Positron emission tomography scans were started with a tracer injection using a multiprogramming syringe pump, which provides a slowly increasing injection volume to achieve steady-state radioactivity for H₂¹⁵O and ¹⁵O₂-HbV scans. The radioactivity concentration of ¹⁵O rapidly achieved equilibrium in the blood and whole brain at about 2 minutes after H₂¹⁵O and ¹⁵O₂-HbV administration, which was stable during the scans. The whole brain mean values of CBF, CBV, and CMRO₂ were 54.3±2.0 mL per 100 g per minute, 4.9±0.4 mL/100 g, and 2.8±0.2 μmoL per g per minute (6.2±0.4 mL per 100 g per minute) in the normal rats, respectively. In the MCAO model rats, all hemodynamic parameters of the infarction area on the occlusion side significantly decreased. The steady-state method with ¹⁵O-labeled HbV is simple and useful to analyze hemodynamic changes in studies with model animals.     

Sleep deprivation attenuates experimental stroke severity in rats

Indirect epidemiological and experimental evidence suggest that the severity of injury during stroke is influenced by prior sleep history. The aim of our study was to test the effect of acute sleep deprivation on early outcome following experimental stroke. Young male Sprague-Dawley rats (n = 20) were subjected to focal cerebral ischemia by reversible right middle cerebral artery occlusion (MCAO) for 90 min. In 10 rats, MCAO was performed just after 6-h of total sleep deprivation (TSD) by “gentle handling”, whereas the other rats served as controls. Neurological function during the first week after stroke was monitored using a battery of behavioral tests investigating the asymmetry of sensorimotor deficit (tape removal test and cylinder test), bilateral sensorimotor coordination (rotor-rod and Inclined plane) and memory (T-maze and radial maze). Following MCAO, control rats had impaired behavioral performance in all tests. The largest impairment was noted in the tape test where the tape removal time from the left forelimb (contralateral to MCAO) was increased by ∼ 10 fold (p < 0.01). In contrast, rats subjected to TSD had complete recovery of sensorimotor performance consistent with a 2.5 fold smaller infarct volume and reduced morphological signs of neuronal injury at day 7 after MCAO. Our data suggest that brief TSD induces a neuroprotective response that limits the severity of a subsequent stroke, similar to rapid ischemic preconditioning.     

Hawthorn extract reduces infarct volume and improves neurological score by reducing oxidative stress in rat brain following middle cerebral artery occlusion

In our present investigation the neuroprotective effect of alcoholic extract of Hawthorn (Crataegus oxycantha) was evaluated against middle cerebral artery occlusion induced ischemia/reperfusion injury in rats. Male Sprague–Dawley rats were pretreated with 100 mg/kg body weight of the extract by oral gavage for 15 days. The middle cerebral artery was then occluded for 75 min followed by 24 h of reperfusion. The pretreated rats showed significantly improved neurological behavior with reduced brain infarct when compared to vehicle control rats. The glutathione level in brain was found to be significantly (p < 0.05) low in vehicle control rats after 24 h of reperfusion when compared to sham operated animals. However, in Hawthorn extract pretreated rats the levels were found to be close to that of sham. Malondialdehyde levels in brain of sham and pretreated group were found to be significantly lower than the non-treated vehicle group (p < 0.05). The nitric oxide levels in brain were measured and found to be significantly (p < 0.05) higher in vehicle than in sham or extract treated rats.     

Auditory orienting and inhibition of return in schizophrenia: A functional magnetic resonance imaging study

Background

The brain mechanisms of cognitive-behavioral therapy (CBT), a highly effective treatment for pediatric obsessive-compulsive disorder (OCD), are unknown. Neuroimaging in adult OCD indicates that CBT is associated with metabolic changes in striatum, thalamus, and anterior cingulate cortex. We therefore probed putative metabolic effects of CBT on these brain structures in pediatric OCD using proton magnetic resonance spectroscopic imaging (¹H MRSI).

Method

Five unmedicated OCD patients (4 ♀, 13.5 ± 2.8) and 9 healthy controls (7 ♀, 13.0 ± 2.5) underwent MRSI (1.5 T, repetition-time/echo-time = 1500/30 ms) of bilateral putamen, thalamus and pregenual anterior cingulate cortex (pACC). Patients were rescanned after 12 weeks of exposure-based CBT. The Children's Yale-Brown Obsessive-Compulsive Scale (CY-BOCS) of OCD symptoms was administered before and after CBT.

Results

Four of 5 patients responded to CBT (mean 32.8% CY-BOCS reduction). Multiple metabolite effects emerged. Pre-CBT, N-acetyl-aspartate + N-acetyl-aspartyl-glutamate (tNAA) in left pregenual anterior cingulate cortex (pACC) was 55.5% higher in patients than controls. Post-CBT, tNAA (15.0%) and Cr (23.9%) in left pACC decreased and choline compounds (Cho) in right thalamus increased (10.6%) in all 5 patients. In left thalamus, lower pre-CBT tNAA, glutamate + glutamine (Glx), and myo-inositol (mI) predicted greater post-CBT drop in CY-BOCS (r = 0.98) and CY-BOCS decrease correlated with increased Cho.

Conclusions

Interpretations are offered in terms of the Glutamatergic Hypothesis of Pediatric OCD. Similar to ¹⁸FDG-PET in adults, objectively measurable regional MRSI metabolites may indicate pediatric OCD and predict its response to CBT.     

Could dietary trans fatty acids induce movement disorders? Effects of exercise and its influence on Na⁺K⁺-ATPase and catalase activity in rat striatum

The influence of trans fatty acids (FA) on development of orofacial dyskinesia (OD) and locomotor activity was evaluated. Rats were fed with diets enriched with 20% soybean oil (SO; n − 6 FA), lard (L; saturated FA) or hydrogenated vegetable fat (HVF; trans FA) for 60 weeks. In the last 12 weeks each group was subdivided into sedentary and exercised (swimming). Brains of HVF and L-fed rats incorporated 0.33% and 0.20% of trans FA, respectively, while SO-fed group showed no incorporation of trans FA. HVF increased OD, while exercise exacerbated this in L and HVF-fed rats. HVF and L reduced locomotor activity, and exercise did not modify. Striatal catalase activity was reduced by L and HVF, but exercise increased its activity in the HVF-fed group. Na⁺K⁺-ATPase activity was not modified by dietary FA, however it was increased by exercise in striatum of SO and L-fed rats. We hypothesized that movement disorders elicited by HVF and less by L could be related to increased dopamine levels in striatum, which have been related to chronic trans FA intake. Exercise increased OD possibly by increase of brain dopamine levels, which generates pro-oxidant metabolites. Thus, a long-term intake of trans FA caused a small but significant brain incorporation of trans FA, which favored development of movement disorders. Exercise worsened behavioral outcomes of HVF and L-fed rats and increased Na⁺K⁺-ATPase activity of L and SO-fed rats, indicating its benefits. HVF blunted beneficial effects of exercise, indicating a critical role of trans FA in brain neurochemistry.     

Significance of minor traumatic lesions in focal head injuries

We investigated the significance of minor traumatic lesions associated with focal head injuries. Patients included in the study were admitted between January 2003 and December 2007 and had sustained a head injury with focal injury and undergone MRI examination including T2^∗-weighted imaging. Patients were divided into two groups: (i) a T2^∗-positive group - those who had hypointense lesions at sites other than the original injury site as shown by T2^∗-weighted MRI (n = 12); and (ii) a control group without hypointense lesions at sites other than the original injury (n = 25). The median Glasgow Outcome Scale score was significantly lower in the T2^∗-positive group (median = 4; range = 4-5) than in the control group (median = 5; range = 4-5; p = 0.003). We conclude that patients with a focal head injury and minor traumatic lesions are likely to have a poorer prognosis than patients without additional minor traumatic lesions.     

Stroke penumbra defined by an MRI-based oxygen challenge technique: 2. Validation based on the consequences of reperfusion

Magnetic resonance imaging (MRI) with oxygen challenge (T₂^* OC) uses oxygen as a metabolic biotracer to define penumbral tissue based on CMRO₂ and oxygen extraction fraction. Penumbra displays a greater T₂^* signal change during OC than surrounding tissue. Since timely restoration of cerebral blood flow (CBF) should salvage penumbra, T₂^* OC was tested by examining the consequences of reperfusion on T₂^* OC-defined penumbra. Transient ischemia (109±20 minutes) was induced in male Sprague-Dawley rats (n=8). Penumbra was identified on T₂^*-weighted MRI during OC. Ischemia and ischemic injury were identified on CBF and apparent diffusion coefficient maps, respectively. Reperfusion was induced and scans repeated. T₂ for final infarct and T₂^* OC were run on day 7. T₂^* signal increase to OC was 3.4% in contralateral cortex and caudate nucleus and was unaffected by reperfusion. In OC-defined penumbra, T₂^* signal increased by 8.4%±4.1% during ischemia and returned to 3.25%±0.8% following reperfusion. Ischemic core T₂^* signal increase was 0.39%±0.47% during ischemia and 0.84%±1.8% on reperfusion. Penumbral CBF increased from 41.94±13 to 116.5±25 mL per 100 g per minute on reperfusion. On day 7, OC-defined penumbra gave a normal OC response and was located outside the infarct. T₂^* OC-defined penumbra recovered when CBF was restored, providing further validation of the utility of T₂^* OC for acute stroke management.     

The cerebral redox state in cats during severe insulin induced hypoglycemia

The cerebral metabolic state was studied in cats during insulin-induced hypoglycemia and the recovery after glucose infusion. Changes in the redox state of nicotinamide adenine dinucleotide (NAD) were monitored from the surface of the exposed cerebral cortex using microfluorometry. After insulin injection blood glucose fell from 6.85 μmol/ml to 0.45 μmol/ml at EEG isoelectricity and was accompanied by an oxidation of NADH⁺. Upon intravenous glucose infusion EEG activity rapidly returned and NAD became more reduced. The oxidation of NADH⁺ during severe hypoglycemia demonstrated that the in vivo redox state of mitochondria behave in a similar manner as isolated mitochondria when reducing equivalents become limiting to the respiratory chain.     

Ischemia-induced alterations in oxidative "recovery" metabolism after spreading cortical depression in situ.

Surface electrical stimulation was used to provoke direct cortical responses, spreading cortical depression, and concomitant increases in oxidative metabolic activity in cat neocortex in situ during and after short periods of incomplete and complete ischemia. These metabolic changes were recorded continuously in the intact tissue by monitoring the fluorescence of NADH (as NAD⁺ does not fluoresce under these optical conditions). Ischemic effects were unidirectional and progressive with the degree of perfusion decrease. Incomplete ischemia was accompanied by decreased excitability and amplitude of metabolic responses to stimulation. During incomplete ischemia when the NADH:NAD⁺ ratio was increased, the brain continued to respond to spreading cortical depression with oxidation of NADH indicating that the capability for increased respiration still resided within the tissue. In cats that survived complete ischemia, these reaction rates became unchanged from control. In other cats, the rate of NAD⁺ re-reduction after spreading depression continued to decrease and death inevitably followed. These findings confirm that short ischemic periods produce alterations in oxidative metabolic capabilities indicative of respiratory uncoupling, resulting in decreased excitability and decreased capacity to respond to increased metabolic demand.     

Microbore liquid chromatography with UV detection to study the in vivo passage of compound 21, a non-peptidergic AT₂ receptor agonist, to the striatum in rats

A microbore liquid chromatography method coupled to UV detection was developed and validated in order to monitor the passage of compound 21 (C21), a non-peptide angiotensin II type 2 receptor agonist, to the striatum of rats. For this purpose, sampling from the striatum was performed using the in vivo microdialysis technique. Separations were performed on a C₁₈ microbore (1 mm i.d.) column using gradient elution. The retention time for C21 was found to be 6.3 min. The calibration curve was linear between 10 and 200 ng/ml with a correlation coefficient ≥0.999. The limit of detection and the limit of quantification were 3 and 10 ng/ml respectively. The intra-day and the inter-day precision (RSD%) ranged between 0.5 and 4.6% with an average recovery of 101.5 ± 10.0% (mean ± SD, n = 15). In vivo experiments were performed on rats to measure the concentration of C21 in striatal dialysates after intraperitoneal (10 or 50 mg/kg) or intravenous injection (10 mg/kg or 20 mg/kg) of C21 and suggest minimal passage of the compound to the striatum.