Ocular and cerebellar defects in zebrafish induced by overexpression of the LIM domains of the islet-3 LIM/homeodomain protein.

Islet-3 is an LIM/homeodomain protein that is expressed specifically in the eyes and the presumptive tectum in the central nervous system of zebrafish (Danio rerio) embryos. Overexpression of the protein (LIM(Isl-3)) consisting only of the Islet-3 LIM domains in embryos specifically prevented formation of the optic vesicles; caused abnormal termination of the expression of wnt1, engrailed2, and pax2 in the mesencephalic and metencephalic region between 14 hr and 20 hr postfertilization; and severely impaired morphogenetic movement in this region between 20 hr and 26 hr, which should normally lead to formation of the cerebellar primordium. Such defects were all rescued by simultaneous overexpression of Islet-3, suggesting that LIM(Isl-3) acted as a specific dominant-negative variant of Islet-3. These data, combined with the results of mosaic analyses, suggest that Islet-3 is activated by putative LIM-binding cofactors and functions to promote evagination of the optic vesicles and to maintain reciprocal interaction between the mesencephalon and the mesencephalic-metencephalic boundary essential for normal development of this region.

Magnetic resonance imaging in the canine double-haemorrhage subarachnoid haemorrhage model.

In this study, we investigated T2 weighted imaging (T2WI) and T2 values of the cortex, thalamus and cerebrospinal fluid (CSF) of the ventricles in the canine double-haemorrhage subarachnoid haemorrhage (DHSAH) model. T2 values in the cortex increased compared to prescan values from 123.07 +/- 18.72 msec on day 2 to 89.43 +/- 1.98 msec on day 7 (p < 0.05). A trend toward a temporal increase in T2 values was observed in the thalamus, but did not reach significance. The T2 values of the ventricular CSF increased by 102.2% on day 2 and 159.6% on day 7 compared to prescan values. These changes reached significance (p < 0.05) on day 7. Additionally, the ventricular size increased over the study period. Our data suggest that we can use this model to investigate acute brain injury and normal pressure hydrocephalus (NPH) after SAH.

Anti-apoptotic effect of granulocyte-colony stimulating factor after focal cerebral ischemia in the rat.

We investigated the molecular mechanisms of the anti-apoptotic properties of granulocyte-colony stimulating factor (G-CSF) on neurons and whether G-CSF affects glial cell survival following focal cerebral ischemia in rats. Sprague-Dawley rats were subjected to a transient 90 min middle cerebral artery occlusion (MCAO) by the intraluminal occlusion technique. Rats were treated with either a single dose of G-CSF (50 microg/kg, s.c.) at the onset of reperfusion or G-CSF (50 microg/kg body weight, s.c.) was administered starting at the onset of reperfusion and followed by the administration of the same dose per day for an additional 2 days. Brains were harvested either 24 h, 72 h or 2 weeks after reperfusion for assays of infarct volume, immunohistological studies and Western blot analysis for phosphorylated signal transducer and activator of transcription 3 (pSTAT3), Pim-1, bcl-2, Bax, cytochrome c, cellular inhibitor of apoptosis protein 2 (cIAP2), and cleaved caspase-3 levels. G-CSF significantly reduced infarct volume and ameliorated the early neurological outcome. G-CSF treatment significantly up-regulated pSTAT3, Pim-1, bcl-2 expression, and down-regulated cytochrome c release to the cytosol, Bax translocation to the mitochondria, and cleaved caspase-3 levels in neurons. The activation of the STAT3 pathway was accompanied by increased cIAP2 expression in glial cells. After MCAO, G-CSF treatment increased both neuronal and glial survival by effecting different anti-apoptotic pathways which reflects the multifactorial actions of this drug. These changes were associated with remarkable improvement in tissue preservation and behavioral outcome.

Role of excitatory amino acid-mediated ionic fluxes in traumatic brain injury.

One major event taking place at the moment of traumatic brain injury in neuronal cells is the occurrence of massive ionic fluxes across the plasma membrane, which can be referred to as traumatic depolarization (TD). Unlike spreading depression, TD can occur over wide brain areas simultaneously. Furthermore, recovery from TD often takes far longer than recovery from ionic perturbation elicited by the passage of a single wave of spreading depression. Neuronal cell damage caused by ischemic brain injury is also initiated by massive ionic fluxes, termed anoxic depolarization. The occurrence of similar ionic events in these two forms of brain injury may account for the genesis of diffuse ischemia-like damage without actual episodes of hypoxia or ischemia in traumatic brain injury. We review the data indicating that excitatory amino acids (EAA) may play a vital role in producing TD, and that such EAA-mediated ionic perturbation is responsible for a number of posttraumatic events including subcellular metabolic dysfunction and cellular responses such as microglial activation and astrocytic transformation. TD may represent one of the most important mechanisms of diffuse neuronal cell dysfunction and damage associated with traumatic brain injury.

Response of regional cerebral blood flow and oxygen metabolism to thalamic stimulation in humans as revealed by positron emission tomography.

To clarify functional neural pathways originating from the thalamic nucleus ventralis posterolateralis (VPL) in humans, the responses of regional CBF (rCBF) and regional CMRO2 (rCMRO2) to VPL stimulation were investigated by positron emission tomography in five patients who had undergone chronic implantation of electrodes into the VPL for therapeutic purposes. Measurement of rCBF and rCMRO2 under continuous inhalation of C15O2 and 15O2 by steady-state methods revealed significant increases of rCBF and rCMRO2 in the frontal, postcentral, and thalamic regions. The increases in rCBF and rCMRO2 of the postcentral regions were clearly predominant in the stimulated hemisphere insofar as the stimulation produced moderate paresthesia in restricted areas of the body. These results indicate that the VPL relays peripheral somatosensory information, which has previously been demonstrated to be transmitted to the frontal as well as postcentral regions.

Pharmacological classification of central post-stroke pain: comparison with the results of chronic motor cortex stimulation therapy.

In an attempt to clarify the neurochemical background of central post-stroke pain and to undertake a pharmacological analysis, the basic pharmacological characteristics of this intractable pain syndrome were investigated by the morphine, thiamylal and ketamine tests. In addition, the correlation between the pharmacological characteristics and the effects of chronic motor cortex stimulation therapy was examined. The study employed 39 central post-stroke pain patients who had intractable hemibody pain associated with dysesthesias, and radiologically demonstrated lesions in the thalamic area (thalamic pain, n = 25) or suprathalamic area (suprathalamic pain, n = 14). The pharmacological evaluations showed that definite pain reduction occurred in eight of the 39 cases (20.5%) by the morphine test, in 22 of the 39 cases (56.4%) by the thiamylal test, and in 11 of 23 cases (47.8%) by the ketamine test. Based on these pharmacological assessments, there was no obvious difference between thalamic and suprathalamic pain. A comparison of the long-term follow-up results of chronic motor cortex stimulation therapy revealed that thiamylal and ketamine-sensitive and morphine-resistant cases displayed long-lasting pain reduction with chronic motor cortex stimulation therapy, whereas the remaining cases did not show good results. We conclude that pharmacological classification of central post-stroke pain by the morphine, thiamylal and ketamine tests could be useful for predicting the effects of chronic motor cortex stimulation therapy. It has recently been suggested that excitatory amino acids may be involved in the development of central post-stroke pain. However, the fact that only 23 of the present 39 cases (59.0%) of thalamic and suprathalamic pain were sensitive to the thiamylal or ketamine test reflects the complex pharmacological background and the difficulties associated with treating central post-stroke pain.

Effects of indeloxazine on hippocampal CA1 pyramidal cell damage following transient cerebral ischemia in the gerbil.

The effects of indeloxazine on the ischemia-induced death of hippocampal CA1 pyramidal cells following transient cerebral ischemia were examined in the mongolian gerbil. Increased survival of CA1 pyramidal cells was demonstrated in animals pre- and post-treated with indeloxazine. Increased survival of CA1 pyramidal cells was, however, not demonstrated in animals post-treated but not pre-treated with indeloxazine. A previous study has demonstrated that indeloxazine increases the glucose and adenosine triphosphate (ATP) contents in the brain probably through an enhanced capability of oxidative phosphorylation. It has been reported that increases in the glucose and ATP contents in the brain before ischemia delay the onset of massive ionic fluxes during ischemia. The delay in onset of this ionic event may help to protect these cells from death. The present data suggest that energy state before ischemia may play an important role in the protective effect of indeloxazine.

Quantitative evaluation of hemiparesis with corticomyographic motor evoked potential recorded by transcranial magnetic stimulation.

Corticomyographic motor evoked potentials (MEP) activated by transcranial magnetic stimulation of the motor cortex provide clinicians with an opportunity to evaluate corticospinal motor systems quantitatively and noninvasively. Threshold, amplitude, and latency of the corticomyographic MEP, however, are variable between subjects mainly because current directions and intensities induced by magnetic stimulation cannot be determined precisely due to anatomical variations of subjects. The variability of corticomyographic MEPs has limited the use of corticomyographic MEP for evaluating mild changes in corticospinal motor function. In the present study, we used an internal standard to assess hemiplegia, expressing relative amplitude, latency, and threshold of responses on the paretic side as a function of responses elicited from the intact side (%MEP). Neurological function of paretic muscles, as determined by a muscle maneuver test (MMT), clearly correlated to %MEP threshold, amplitude, and latency. Since corticomyographic MEP are similar when recorded from symmetrical sites on two extremities of normal subjects, %MEP provided a sensitive measure of mild hemiparesis. The %MEP approach revealed abnormal MMT scores of 3 or 4 more frequently than did standard MEP approaches. %MEP amplitude was more sensitive to mild hemiparesis than %MEP latency or %MEP threshold. Since magnetic stimulation with a safe intensity range cannot reliably produce corticomyographic MEP in severely paretic muscles with MMT scores of 2 or less, the MEP appears to be most useful for evaluating mild hemiparesis. This technique should expand significantly the clinical usefulness of corticomyographic MEP in neurosurgical practice.

Spinal cord responses to feline transcranial brain stimulation: evidence for involvement of cerebellar pathways.

The physiological characteristics of spinal cord responses recorded from the spinal epidural space of the cat to transcranial brain stimulation were studied, in comparison with the spinal cord responses to direct stimulation of the motor cortex or cerebellum. The conduction velocity of the initial wave of the responses to transcranial brain stimulation (122.3 +/- 16.3 m/sec mean +/- SD, n = 5) was much faster than the conduction velocity of the initial wave of the responses to motor cortex stimulation (68.3 +/- 14.7, n = 5) and similar to the conduction velocity of the initial wave of the responses to cerebellar stimulation (120.2 +/- 16.2, n = 5). Furthermore, the conduction velocity of any component in the subsequent polyphasic waves at any intensity was not similar to the conduction velocity of the initial wave of the responses to motor cortex stimulation. All components of the responses to motor cortex stimulation disappeared after intercollicular transection. In contrast, the initial wave of the responses to cerebellar stimulation and transcranial brain stimulation remained unaffected by intercollicular transection. The initial wave caused by anodal transcranial brain stimulation was eliminated by ablation of the cerebellum. However, cathodal transcranial brain stimulation sometimes can produce an initial wave that can be eliminated only by transection at the medullospinal junction. The initial wave of the responses to cerebellar stimulation was largest in amplitude when the vicinity of the dentate nucleus was stimulated. These results suggest that responses to activation of the cerebellum, rather than corticospinal neurons arising from the motor cortex, represent a major component of the spinal cord responses to transcranial brain stimulation in cats. The data obtained indicate that it is difficult to activate the motor cortex selectively by transcranial brain stimulation in cats.

Electrophysiological evidence favoring intracaudate axon collaterals of GABAergic caudate output neurons in the cat.

Stimulation of the entopeduncular nucleus in the cat was shown to evoke inhibitory responses with a short onset-latency in the caudate nucleus isolated from its afferents. These inhibitory responses are shown to be GABAergic, and some of them are suggested to be monosynaptic in nature.

Excitatory amino acid antagonist administered via microdialysis attenuates lactate accumulation during cerebral ischemia and subsequent hippocampal damage.

Our previous studies have shown that kynurenic acid (KYN), a broad-spectrum antagonist of excitatory amino acids (EAAs), administered in situ through a dialysis probe can delay the massive ionic fluxes in the rat hippocampus during cerebral ischemia. The present experiments demonstrated that the same procedure attenuates the increase in extracellular concentration of lactate ([lactate]e) during ischemia as measured by microdialysis. This finding suggests that the lactate accumulation is partially caused by a sudden increase in energy demand due to the rapid ionic fluxes through EAA-coupled ion channels. This inference is consistent with the hypothesis that the earlier ionic event during ischemia is a cause of energy depletion, rather than the result merely of energy failure. The present experiments also revealed that KYN administered by the same procedure attenuates death of hippocampal CA1 pyramidal cells after 5-min transient ischemia in gerbils. Since lactate accumulation is likely to be an important factor affecting cell viability, the protective effect of KYN may be attributable, in part, to inhibition of lactate accumulation.

Temporal pattern of survival and dendritic growth of fetal hippocampal cells transplanted into ischemic lesions of the adult rat hippocampus.

Cell suspensions obtained from the fetal hippocampus were transplanted into the adult rat hippocampus at 1 or 4 weeks after transient forebrain ischemia. Only when the ischemia induced death of most of the CA1 pyramidal cells of the host hippocampus and transplantation was performed at 1 week after the ischemia, did a large number of transplanted cells survive and the most extensive dendritic growth was demonstrated by microtubule-associated protein 2 immunohistochemistry. The dendrites of the cells located in the ventral part were oriented ventrally, lining up similarly to the parallel arrangements of apical dendrites of normal CA1 pyramidal cells. These findings suggest that certain forms of trophic factors, which appear to occur in association with the presence of free terminals of afferent fibers during the earlier period after ischemic insult, are involved in the survival of and dendritic growth from transplanted hippocampal cells.

Long lasting suppression of firing of cortical neurons and decrease in cortical blood flow following train pulse stimulation of the locus coeruleus in the cat.

Single neuron activity and local cerebral blood flow were recorded simultaneously in the same spot of the gyrus proreus in cats. Train pulse stimulation (10-20 Hz, 30 sec) of the ipsilateral locus coeruleus induced long lasting suppression of firing in up to 78% of neurons and decrease in local flow, which lasted 1.9-5.6 min and 3.8-6.5 min, respectively. Single pulse stimulation evoked inhibition of firing in 55% of the neurons investigated.

Evidence for involvement of the frontal cortex in pain-related cerebral events in cats: increase in local cerebral blood flow by noxious stimuli.

Noxious stimuli were shown to induce a remarkable increase in local cerebral blood flow restricted to the forepart of the cerebral hemispheres bilaterally anterior to the posterior sigmoid gyrus in cats. This increase in local cerebral blood flow was averted by lesions in the bilateral ventromedial thalamus and attenuated by pretreatment with an intraventricular injection of 6-hydroxydopamine.

Early cellular swelling during cerebral ischemia in vivo is mediated by excitatory amino acids released from nerve terminals.

This study demonstrates ischemic cellular swelling in vivo detected as changes in the concentration of 14C-sucrose pre-perfused into the extracellular space (ECS) as an ECS marker. Microdialysis was utilized as a means of perfusion and measurement of the extracellular concentration of 14C-sucrose ([14C-sucrose]e). Concomitant with an abrupt increase in [K+]e at 1-3 min following the ischemia induction, [14C-sucrose]e was also rapidly elevated. Since sucrose is not taken up by either cells or capillaries, the absolute amount of 14C-sucrose in the ECS must be unchanged. The increase therefore appears to represent a relative decrease in water volume in the ECS resulting from a movement of water into the cells, i.e. cellular swelling. Ca(2+)-free perfusate containing Co2+, which has been shown to block excitatory amino acid release during cerebral ischemia, significantly delayed the increase in [14C-sucrose]e and [K+]e. Kynurenic acid, a broad-spectrum antagonist of excitatory amino acids, administered in situ through the dialysis probe also significantly delayed the increase in [14C-sucrose]e and [K+]e. These findings indicate that the early cellular swelling occurring during cerebral ischemia is a result of massive ionic fluxes mediated by excitatory amino acids which are released by a Ca(2+)-dependent exocytotic process from the nerve terminals.

Calcium-dependent component of massive increase in extracellular potassium during cerebral ischemia as demonstrated by microdialysis in vivo.

This study characterizes the physiological features and limitations of K(+)-free dialysis to detect changes in extracellular concentration of K+ ([K+]e) in the rat hippocampus in vivo. It also demonstrates the effects of Ca(2+)-free perfusate containing Co2+ or Mg2+, which blocks Ca2+ entry into the presynaptic nerve terminal, on the abrupt increase in [K+]e detected by this technique during cerebral ischemia. K(+)-free dialysis for 40 min caused no significant changes in the baseline [K+]e. In contrast, Ca(2+)-free dialysis for 40 min significantly reduced the extracellular Ca2+ concentration. Under this condition, together with addition of Co2+ or Mg2+ to the perfusate, the increase in [K+]e was delayed, and a delay in reaching the maximum level was observed in a dose-dependent manner. These results are consistent with the hypothesis that the initial increase in [K+]e during cerebral ischemia is related to the Ca(2+)-dependent exocytotic release of neurotransmitters from depolarized nerve terminals.

Inhibition of the early phase of free fatty acid liberation during cerebral ischemia by excitatory amino acid antagonist administered by microdialysis.

In order to determine the role of excitatory amino acids (EAAs) in free fatty acid (FFA) liberation during cerebral ischemia, we examined the effect of in situ administration of kynurenic acid, a broad-spectrum antagonist of EAA receptors, by microdialysis on the increase in FFA levels during ischemia in the rat hippocampus. A transient rapid increase in FFA levels, superimposed on a continued slow increase, was observed beginning at 1-2 min after ischemia induction. The early rapid increase in FFAs was profoundly inhibited by kynurenic acid, suggesting that EAAs are critically involved in the early phase of FFA liberation. Development of massive ionic shifts during cerebral ischemia can be delayed for several minutes by kynurenic acid administered by the same procedure, suggesting a vital role for EAAs in the early appearance of anoxic depolarization. The observed inhibition of early FFA liberation may thus be attributable to the delay in development of massive ionic shifts and resultant neurotransmitter release which may activate phospholipase A2 and C.

Inhibition of rapid potassium flux during cerebral ischemia in vivo with an excitatory amino acid antagonist.

Previous studies have demonstrated that microdialysis is capable of detecting an abrupt and massive increase in extracellular K+ concentration ([K+]e) and a concomitant increase in extracellular concentration of excitatory amino acids (EAAs) during cerebral ischemia in the rat hippocampus in vivo. Following in situ administration of kynurenic acid (KYN), a broad-spectrum antagonist of EAAs, through the dialysis probe (5-10 mM), a delay in reaching the maximum level of increased [K+]e was observed in a dose-dependent manner. The initial component of the rapid increase in [K+]e appears to be mediated by EAAs released from nerve terminals.

Propentofylline administered by microdialysis attenuates ischemia-induced hippocampal damage but not excitatory amino acid release in gerbils.

Systemic administration of propentofylline (PPF), an adenosine uptake inhibitor, has been demonstrated to protect CA1 pyramidal cells from death following transient cerebral ischemia in gerbils. In order to examine the direct effects of this inhibitor, we tested whether or not PPF administered into the hippocampus in situ through a microdialysis probe could attenuate ischemia-induced excitatory amino acid (EAA) release and prevent subsequent death of CA1 pyramidal cells in the gerbil. The EAA release and death of CA1 pyramidal cells observed in the hippocampus were compared with those in the contralateral hippocampus of the same animal into which vehicle alone was administered. The results indicated that pre- as well as post-treatments with PPF inhibited the death of CA1 pyramidal cells after 5-min ischemia in a dose-dependent manner, but did not significantly alter the EAA release during ischemia and reperfusion in the same animals. While the neuroprotective effect of PPF against ischemic damage has commonly been ascribed to attenuation of EAA release during ischemia, other actions of adenosine such as those influencing the synaptic responses, neuronal excitation, and local cerebral circulation, or as yet unidentified actions may be involved in the observed neuroprotective effects of PPF.

Calcium-dependent glutamate release concomitant with massive potassium flux during cerebral ischemia in vivo.

The changes in extracellular glutamate ([Glu]e) and potassium ([K+]e) in the rat hippocampus during cerebral ischemia were determined simultaneously by microdialysis in vivo. Biphasic increases in [Glu]e, i.e. an earlier rapid increase concomitant with an abrupt increase in [K+]e followed by a later slow increase, were observed. Dialysis with Ca(2+)-free perfusate containing Co2+ blocked the earlier rapid increase completely but the later slow increase only partially. These findings suggest that Ca(2+)-dependent exocytotic release from the presynaptic nerve terminals is involved predominantly in the earlier rapid increase in [Glu]d. The later slow increase in [Glu]d may be due in part to a breakdown of membrane function resulting from several causes, including a loss of the electrogenic component of the glutamate gradients across the plasma membrane, and a loss of function of the glutamate uptake system.