Significant shrinkage of extracellular space during global cerebral ischemia: differences in gray and white matter ischemia.

Brain extracellular space (ECS) provides an important microenvironment for neurons and glial cells. In the present study, we investigated differences in ischemic changes of ECS in gray and white matter during global ischemia in cats (n = 8). Diffusion capacity of tetramethylammonium ion (TMA+, 74.1Da) was evaluated by measuring extracellular TMA+ concentration applied via microdialysis. In both gray and white matter, TMA+ concentration significantly increased within 10 minutes after the induction of ischemia, and sustained during 120 min of ischemia. The increase, however, was slower and smaller in white matter than in gray matter. In other two animals, volume fraction and tortuosity of ECS were determined by iontophoresis during global ischemia. After 30 min of ischemia induction, volume fraction was decreased and tortuosity was increased in both gray and white matter. In white matter, decrease in volume fraction and increase in tortuosity were smaller than in gray matter. The present study demonstrates that cerebral ischemia induces not only significant shrinkage of ECS volume but also restricts molecular diffusion within. Smaller changes of diffusion capacity within white matter ECS during ischemia may be relevant for lower ischemic vulnerability of the region.

Semiquantitative analysis of corpus callosum injury using magnetic resonance imaging indicates clinical severity in patients with diffuse axonal injury.

OBJECTIVE: To evaluate the hypothesis that the extent of corpus callosum injury indicates the depth of shearing lesions in the central brain structure and therefore relates to the clinical severity of diffuse axonal injury. METHODS: A simple and objective procedure for semiquantitative analysis of magnetic resonance images (MRI)-the maximum signal intensity ratio (MSIR)-was employed prospectively in 21 patients with diffuse axonal injury but without apparent injury to the ventral pons. All were diagnosed using serial combination MRI scans of fluid attenuated inversion recovery (FLAIR) and T2* weighted gradient echo imaging during the initial two weeks after the injury. The signal intensity ratio between the two regions of interest-the corpus callosum and the normal appearing ventral pons-was calculated serially in mid-sagittal and parasagittal FLAIR image sections in each patient. The MSIR during the study period was determined as a semiquantitative index of corpus callosum injury in each patient. The correlations between MSIR and the duration of unconsciousness, Glasgow outcome scale at six months, and the presence of apparent midbrain injury were investigated. RESULTS: The mean (SD) MSIR value was 1.12 (0.18) at 7.4 (3.1) days after the injury (n = 21). MSIR correlated strongly with the duration of unconsciousness (n = 19, R(2) = 0.74, p < 0.0001), and was higher in patients with both an unfavourable GOS outcome (p = 0.020) and apparent midbrain injury (p < 0.001). CONCLUSIONS: MSIR, which is a simple and objective procedure for semiquantitative analysis of corpus callosum damage in diffuse axonal injury, correlated with clinical severity. A high MSIR value may indicate the presence of concomitant midbrain injury.

Implantation of a reservoir for refractory chronic subdural hematoma.

OBJECTIVE: Recurrence of chronic subdural hematoma is not rare. Among patients who experience recurrence, severe background disease may adversely influence the prognosis of chronic subdural hematoma. We treated patients with these refractory hematomas with an Ommaya cerebrospinal fluid (CSF) reservoir and analyzed the effectiveness of the treatment. METHODS: Sixteen patients with refractory chronic subdural hematoma were studied. These patients had severe diseases that adversely influenced the clinical course of chronic subdural hematoma, including cerebral infarction, liver cirrhosis, thrombocytopenia, severe Parkinsonism, severe heart disease, psychiatric disease, and spinocerebellar degeneration. All patients were treated initially in the standard fashion: evacuation of the hematoma followed by irrigation and drainage of the hematoma cavity. In each patient, an Ommaya CSF reservoir was implanted after the hematoma recurred. Whenever the volume of the hematoma either decreased very slowly or increased, the reservoir was punctured. RESULTS: The hematoma size decreased to less than 3 mm a median of 60 days after introduction of the reservoir. Postoperatively, 13 patients returned to their condition before the onset of hematoma. One patient died of myocardial infarction, and two patients with Parkinson's disease could not maintain their previous functional level; both remained in a partially dependent state. Complications consisted of minor bleeding in two patients and occlusion of the reservoir in two other patients. CONCLUSION: By use of this method, reoperation was avoided and the patients were mobile early in the postoperative period. This method was suitable for refractory chronic subdural hematoma accompanied by severe disease that adversely influenced the clinical course.

Adenosine in relation to calcium homeostasis: comparison between gray and white matter ischemia.

In vitro studies suggest that adenosine may attenuate anoxic white matter damage as an intrinsic protective substance. The authors investigated ischemic alterations of purines in relation to tissue depolarization and extracellular calcium and amino acid concentrations in vivo using microdialysis and ion-selective electrodes in cortical gray and subcortical white matter of 10 cats during 120 minutes of global brain ischemia. Immediately on induction of ischemia, regional cerebral blood flow ceased in all cats in both gray and white matter. The direct current potential rapidly decreased, the decline being slower and shallower in white matter. Extracellular calcium levels decreased in gray matter. In contrast, they first increased in white matter and started to decrease below control levels only after approximately 30 minutes. Adenosine levels transiently increased in both tissue compartments; the peak was delayed by 30 minutes in white matter. Thereafter, levels declined faster in gray than in white matter and remained elevated in the latter tissue compartment. Inosine and hypoxanthine elevations were progressive in both regions but smaller in white matter. Levels of gamma-aminobutyric acid, another putatively protective agent, steadily increased, starting immediately in gray matter and delayed by almost 1 hour in white matter. The delayed and prolonged accumulation of adenosine correlates with a slower adenosine triphosphate breakdown in white matter ischemia and may result in protection of white matter by suspending cellular calcium influx.

Induction of aquaporin-4 water channel mRNA after focal cerebral ischemia in rat.

Aquaporin-4 (AQP4) is a member of a water-selective channel aquaporin-family and mainly expressed in the several structures of the brain and in the collecting duct of the kidney. Here we show its functional involvement in the water homeostasis of the ischemic brain. The expression of AQP4-mRNA is increased in the peri-infarcted cortex during the observation period ( approximately 7 days) after MCA-occlusion, maximally on day 3. The change corresponds to the generation and resolution of brain edema monitored by MRI. The signals for the mRNA are predominantly observed in glial cells in the molecular and outer granular layer of the peri-infarcted cortex. These results indicate that AQP4 plays a role in post-ischemic edema formation.

Brain-derived neurotrophic factor blocks long-term depression in solitary neurones cultured from rat visual cortex.

1. To address questions of whether long-term depression (LTD) in the visual cortex is expressed in pre- or postsynaptic sites, whether brain-derived neurotrophic factor (BDNF) exerts its LTD-blocking action without involvement of GABAergic inhibition, and whether the action of BDNF is pre- or postsynaptic, we observed excitatory postsynaptic currents (EPSCs) from solitary neurones cultured on glial microislands. In this preparation GABAergic inhibition is not involved and a group of synapses (autapses) which generate evoked EPSCs is thought to be the same as those generating spontaneous EPSCs. 2. A short depolarising voltage step to the soma generated Na+ spikes which were followed by autaptic EPSCs. When this somatic activation was paired with prolonged depolarisation for 100 ms to -30 mV and repeated at 1 Hz for 5 min, LTD was induced in all of the nine cells tested. Then, the frequency of spontaneous EPSCs decreased, but the amplitude did not change, suggesting that the site of LTD expression is presynaptic. 3. Application of BDNF at 50 ng ml-1 blocked the depression of evoked EPSCs and the decrease in the frequency of spontaneous EPSCs. An inhibitor for receptor tyrosine kinases, K252a, antagonised the action of BDNF, suggesting an involvement of BDNF receptors, TrkB. 4. These results suggest that BDNF prevents low-frequency inputs from inducing LTD of excitatory synaptic transmission through presynaptic mechanisms in the developing visual cortex.

Decrease in N-acetylaspartate without commensurate accumulation of acetate in focal cerebral ischemia in rat.

N-acetylaspartate (NAA) is a plausible marker of neuronal viability which decreases in a variety of neurodestructive conditions. To elucidate the mechanism that leads to NAA decline in two different types of cerebral ischemia in rats, we simultaneously determined cortical concentrations of NAA and its hydrolytic metabolites, aspartate, and acetate by high-resolution 1H-NMR spectroscopy. NAA decreased almost linearly up to 24 h in both decapitation induced global cerebral ischemia, and in ischemic cortices of focal ischemia. Acetate was increased continuously for up to 24 h of global ischemia, while in focal cerebral ischemia it was increased transiently at 6 h. Aspartate did not show any change in global ischemia, while it was decreased in focal ischemia. Although NAA decreased similarly in the brain with global and focal ischemia, temporal changes of two NAA hydrolytic metabolites were different in each type of ischemia. The present results suggest hydrolytic degradation of NAA may be modified alternatively under each pathophysiologic condition.

Breakdown of calcium homeostasis in relation to tissue depolarization: comparison between gray and white matter ischemia.

In vitro studies suggest that ischemic injury of cerebral white matter is mediated by nonsynaptic cellular mechanisms, such as Ca2+ entry into axons through reversal of the Na+ -Ca2+ exchanger. The authors investigated extracellular Ca2+ concentration in relation to tissue depolarization (direct current potential) in vivo using ion-selective electrodes in cortical gray and subcortical white matter of alpha-chloralose-anesthetized cats during 120 minutes of global cerebral ischemia. On induction of ischemia, regional CBF, as measured by hydrogen clearance, ceased. The direct current potential decreased rapidly within minutes in gray matter and with little time delay in white matter. Extracellular Ca2+ concentration decreased just as quickly in gray matter. In white matter, in contrast, extracellular Ca2+ increased in the first 20 to 30 minutes, and a delayed and much slower decline, compared with gray matter, was observed thereafter, reaching a minimal level only about 60 minutes after occlusion. Our results suggest that smaller and delayed transmembrane shifts of Ca2+ are correlates of delayed ischemic membrane dysfunction in central white matter tracts, which may be explained by a lack of synaptic mechanisms.

Cellular expression of inducible nitric oxide synthase following rat cortical incision and its suppression by hydroxyl radical scavenger, 1,2-bis(nicotinamido)propane.

Cellular expression of an inducible isoform of nitric oxide synthase (iNOS) was studied immunohistochemically 1,3 and 7 days following rat cortical incision. The induction of iNOS was demonstrated almost exclusively in the macrophages accumulated within the incision cavity on day 3. They were significantly reduced in number by the treatment with 1,2-bis(nicotinamido)propane (P < 0.01), suggesting the involvement of hydroxyl radicals in the macrophage activation after cortical injury.

Increase in jugular levels of polymorphonuclear neutrophil elastase in patients with acute cerebral infarction.

To elucidate the involvement of activated polymorphonuclear neutrophil (PMN) proteolytic activity in the pathophysiology of cerebrovascular stroke, we measured internal jugular venous and femoral arterial levels of PMN elastase in 6 patients with acute stroke from the day onset (day 0) to 7 days after onset (day 7). Two patients with cerebral infarction demonstrated markedly elevated of PMN elastase in the jugular blood (730 and 1320 micrograms l-1, respectively), but not in the blood (203 and 205 micrograms l-1, respectively) on day 2. Both patients manifested signs of hemorrhagic infarction early on day 2. On day 4, the increased levels of jugular elastase returned to the normal range (< 250 micrograms l-1 in both patients. In contrast, none of the 4 patients with intracerebral hemorrhage demonstrated increased levels of PMN elastase in either the jugular or the arterial blood from days 0 to 7. The present study demonstrated a temporary but significant increase in jugular PMN elastase in patients with cerebral infarction, especially shortly after hemorrhagic infarction.

Stochastic determination of the chromosomal region responsible for expression of human glial fibrillary acidic protein in astrocytic tumors.

Previous investigators have localized the human glial fibrillary acidic protein (GFAP) gene in the segment 17q21 in chromosome 17. In the present study, we statistically assessed the association between the allelic status in thirty-three microsatellite loci and the immunohistochemical expression of GFAP in twenty human astrocytic tumors. The results demonstrated that the loss of heterozygosity in only one locus (D17S795 located in 17q21.2) was significantly associated with the impaired expression of GFAP (P = 0.0280, Pc = 0.0384 by Fisher's exact test). The adjacent loci located in 17q21.1 and 17q21.3 were not relevant to GFAP expression. Those data suggest that the critical region responsible for GFAP expression (coding sequence and regulatory elements) is located close to the locus D17S795 in the segment 17q21.2.

Hypothermia suppresses nitric oxide elevation during reperfusion after focal cerebral ischemia in rats.

We aimed to investigate effect of temperature on the jugular levels of nitric oxide (NO) at reperfusion after focal cerebral ischemia. Both nitrosyl hemoglobin (HbNO) (2.5 +/- 0.4 microM) and plasma nitrite plus nitrate levels (61 +/- 5 microM) in rats under normothermia (approximately 37 degrees C) after 30 min of reperfusion following 2 h of left middle cerebral artery occlusion were significantly high, compared with sham operated rats (1.3 +/- 0.1 microM, 40 +/- 4 microM, respectively). Both HbNO (1.5 +/- 0.3 microM) and nitrite plus nitrate levels (43 +/- 7 microM) under moderate hypothermia (approximately 32 degrees C) were significantly low, compared with normothermic rats. HbNO (2.8 +/- 0.8 microM) and nitrite plus nitrate levels (65 +/- 8 microM) under mild hyperthermia (approximately 39 degrees C) were not significantly high. These results firstly demonstrated that hypothermia suppresses the elevation in intrajugular NO after cerebral ischemia-reperfusion.

Early nitric oxide increase in depolarized tissue of cat focal cerebral ischaemia.

Tissue nitric oxide (NO) concentration was investigated in relation to ion-homeostasis disturbance in the cat model of focal cerebral ischaemia. An NO electrode, a Ca2+ microelectrode and a laser Doppler probe were applied to the cerebral cortex in the core and periphery of the middle cerebral artery. NO concentration increased by 25.1 +/- 6.3 nM at 5 min in severely ischaemic regions exhibiting anoxic depolarization (n = 5, p < 0.0005). This occurred with no reduction in extracellular Ca2+ concentration and before a massive Ca2+ influx into cells started several minutes later. The NO increase was abolished by NG-nitro-L-arginine treatment (n = 6, p < 0.05) and was absent in regions with no depolarization (n = 5, p < 0.0005). We conclude that the early increase in NO associated with depolarization is achieved by activation of constitutive NO synthase, possibly triggered by intracellular Ca2+ release.

Detection of nitric oxide production and its role in pancreatic ischemia-reperfusion in rats.

Partial ischemia of rat pancreas body and tail was obtained by occlusion of the celiac axis for 1 h after gastrectomy. The plasma level of nitrite plus nitrate in both systemic and portal venous blood after reperfusion was significantly higher than that after sham operation and ischemia alone. The elevation after reperfusion was significantly decreased by NG-nitro-L-arginine methyl ester (L-NAME). Simultaneous administration of L-arginine counteracted the L-NAME-induced decrease in the level of nitric oxide (NO) end products. Generation of NO was further demonstrated by nitrosylhemoglobin detection by electron spin resonance in the blood after reperfusion. On the other hand, the plasma level of lipase, a marker of damage to pancreatic exocrine tissue, was significantly increased after ischemia-reperfusion and further increased by administration of L-NAME. This increase in lipase correlated with a decrease in tissue blood flow in the pancreas. These results suggest that NO is generated during and may have a protective role in ischemia-reperfusion of the rat pancreas.

Generation of nitric oxide and superoxide during reperfusion after focal cerebral ischemia in rats.

We investigated the levels of nitrosyl hemoglobin (HbNO) in rat jugular blood by electron spin resonance (ESR) spectroscopy during and after middle cerebral artery occlusion. The levels of plasma nitric oxide (NO) end products, nitrate plus nitrate, were compared with the levels of HbNO. Small amounts of HbNO were detected in sham-operated rats (n=4) and those subjected to 2 h of occlusion (n=4), whereas nitrite plus nitrate was increased only in the latter (P<0.01; vs.sham). Upon reperfusion after 2 h of occlusion both HbNO and nitrite plus nitrate clearly increased after 15 min (n=4) and 30 min (n=6) reperfusion (P<0.01; vs.occlusion). Administration of superoxide dismutase (5 mg/kg) significantly increased HbNO (P<0.05) but not plasma nitrate plus nitrate (n=5). The increase in HbNO suppressed by administration of NG-nitro-L-arginine methyl ester (20mg/kg; n=4,P<0.01), and this suppression could be reversed by L-arginine (200 mg/kg) (n=4). The present study clearly showed that the L-arginine-NO synthase pathway was activated during reperfusion after focal cerebral ischemia and indicated the involvement of a reaction between NO and superoxide during early reperfusion.

Effects of superoxide dismutase on nitric oxide production during reperfusion after focal cerebral ischemia is rats.

To assess the interaction of nitric oxide (NO) and superoxide during reperfusion after cerebral ischemia, we studied the dose effects of superoxide dismutase (SOD) on the levels of nitrosyl hemoglobin and plasma nitrite + nitrate which were increased at 30 min reperfusion after 2 h middle cerebral artery occlusion in rats. SOD was administered at 10 min before reperfusion. With 1, 5 and 10 mg/Kg of SOD, plasma nitrite + nitrate level was decreased by 25 mg/kg of SOD. The same amount of apo enzyme was without effect. These results suggest effect of superoxide in the NO level released during reperfusion after cerebral ischemia.

Generation of nitric oxide as a rejection marker in rat pancreas transplantation.

In clinical pancreas transplantation, no reliable marker for the early diagnosis of acute rejection has been reported. This is one reason why the graft survival rate of pancreas transplantation alone is much lower than that of other organs, such as hearts, livers, and kidneys. We designed an experiment to investigate acute rejection of pancreas allografts in hyperglycemic rats by measurement of blood glucose levels and nitric oxide (NO) products (nitrite plus nitrate, and nitrosyl hemoglobin). As recipients, Lewis rats were rendered hyperglycemic by intravenous injection of streptozotocin before transplantation. F344 rats were used as donors of pancreas allografts. Lewis rats were also used as donors of syngeneic pancreas grafts. After transplantation, the blood glucose level returned to a normal level and rejection was defined as the recurrence of hyperglycemia. The mean survival time of pancreas allografts was 14 +/- 0.7 days. The plasma level of nitrite plus nitrate in allografted rats peaked on postoperative day 7. Electron spin resonance spectra of NO bound to hemoglobin were detected in the blood from allografted rats with a peak on postoperative day 7, whereas NO bound to hemoglobin was not detected in the blood from recipients of syngeneic grafts at any sampling time. The results show that NO was synthesized in the earlier period than the elevation of the blood glucose level during rejection after pancreas transplantation in rats.

Reaction of microglial cells and macrophages after cortical incision in rats: effect of a synthesized free radical scavenger, (+/-)-N,N'-propylenedinicotinamide (AVS)

Reactive microglial cells and macrophages appear after trauma to the brain. To investigate the accumulation patterns of reactive microglial cells and macrophages after cortical incision, these cells were stained immunohistochemically with anti-ED1 antibody in the brain sections before and 1, 3, 5, and 7 days after incision. And to ascertain the participation of oxygen free radicals in these cellular reactions, a synthesized free radical scavenger, (+/-)-N,N'-propylenedinicotinamide (AVS) was administered in this model. Rats were administered AVS (300 mg/kg, i.p.) 30 min before, 2.5 h and every 24 h after incision (AVS group), while only saline was administered in the same manner as a control (saline group). In the saline group, both reactive microglial cells and macrophages had already appeared on day 1 post-incision. The former continued to increase in number during the following days, whereas the latter increased in number up to day 3 and thereafter decreased. Both the numbers of reactive microglial cells and macrophages were significantly decreased (P < 0.05) in the AVS group on days 5 and 7. The results suggest the participation of oxygen free radicals in the reaction of microglial cells and macrophages in traumatic brain injury.

Increase in plasma nitric oxide end products following rat cortical injury.

The changes in plasma nitric oxide (NO) end products, nitrite (NO2-) and nitrate (NO3-), were studied following cortical injury in rats. At 3 days after stereotactic cortical injury (day 3), plasma NO end products were significantly increased (P < 0.01), and decreased by day 7. This increase on day 3 was inhibited by a selective inhibitor of inducible NO synthase (NOS), aminoguanidine (100 mg/kg, i.p. on days 1 and 2, P < 0.001). The present study first demonstrated the temporary increase in plasma NO end products, which is attributable to the inducible NOS activation after cerebral injury.