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S100B is an astrocyte-derived protein that can act through the receptor for advanced glycation endproducts (RAGE) to mediate either "trophic" or "toxic" responses. Its levels increase in many neurological conditions with associated microvascular dysregulation, such as subarachnoid hemorrhage (SAH) and traumatic brain injury. The role of S100B in the pathogenesis of microvasculopathy has not been addressed. This study was designed to examine whether S100B alters pial arteriolar vasodilating function. Rats were randomized to receive (1) artificial cerebrospinal fluid (aCSF), (2) exogenous S100B, and (3) exogenous S100B+the decoy soluble RAGE (sRAGE). S100B was infused intracerebroventricularly (icv) using an osmotic pump and its levels in the CSF were adjusted to achieve a concentration similar to what we observed in SAH. After 48 h of continuous icv infusion, a cranial window/intravital microscopy was applied to animals for evaluation of pial arteriolar dilating responses to sciatic nerve stimulation (SNS), hypercapnia, and topical suffusion of vasodilators including acetylcholine (ACh), s-nitroso-N-acetyl penicillamine (SNAP), or adenosine (ADO). Pial arteriolar dilating responses were calculated as the percentage change of arteriolar diameter in relation to baseline. The continuous S100B infusion for 48 h was associated with reduced responses to the neuronal-dependent vasodilator SNS (p0.05) and the endothelial-dependent vasodilator ACh (p0.05), compared to controls. The inhibitory effects of S100B were prevented by sRAGE. On the other hand, S100B did not alter the responses elicited by vascular smooth muscle cell-dependent vasodilators, namely hypercapnia, SNAP, or ADO. These findings indicate that S100B regulates neuronal and endothelial dependent cerebral arteriolar dilation and suggest that this phenomenon is mediated through RAGE-associated pathways.

Aneurysmal subarachnoid hemorrhage (SAH) is a potentially devastating clinical problem. Despite advances in the diagnosis and treatment of SAH, outcome remains unfavorable. An increased inflammatory state, one that is characterized by enhanced leukocyte trafficking has been reported to contribute to neuronal injury in association with multiple brain insults, including hemorrhagic and ischemic stroke. This study was designed to investigate, in rats, the neuropathologic consequences of heightened leukocyte trafficking following SAH, induced via endovascular perforation of the anterior cerebral artery. Experiments focused on the initial 48 h post-SAH and sought to establish whether blockade of vascular adhesion protein-1 (VAP-1), with LJP-1586, was able to provide dose-dependent neuroprotection. Treatment with LJP-1586 was initiated at 6h post-SAH. An intravital microscopy and closed cranial window system, that permitted examination of temporal patterns of rhodamine-6G-labeled leukocyte adhesion/extravasation, was used. Effects of LJP-1586 on neurologic outcomes and leukocyte trafficking at 24 h and 48 h post-SAH were examined. In VAP-1-inhibited vs control rats, results revealed a significant attenuation in leukocyte trafficking at both 24 h and 48 h after SAH, along with an improvement in neurologic outcome. In conclusion, our findings support the involvement of an amplified inflammatory state, characterized by enhanced leukocyte trafficking, during the first 48 h after SAH. VAP-1 blockade yielded neuroprotection that was associated with an attenuation of leukocyte trafficking and improved neurologic outcome.

We previously demonstrated that chronic hyperglycemia has a detrimental influence on neurovascular coupling in the brain—an effect linked to an alteration in the protein kinase C (PKC)-mediated phosphorylation pattern. Moreover, the activity of PKC was increased, in diabetic rat brain, in a tissue fraction composed primarily of the superficial glia limitans and pial vessels, but trended toward a decrease in cerebral cortical gray matter. However, that study did not examine the expression patterns of PKC isoforms in the rat brain. Thus, , in a rat model of streptozotocin (STZ)-induced chronic type 1 diabetes mellitus (T1DM), and in non-diabetic (ND) controls, two hypotheses were addressed. First, chronic T1DM is accompanied by changes in the expression of PKC-α, βII, γ, δ, and ε. Second, those changes differ when comparing cerebral cortex and glio-pial tissue. In addition, we analyzed the expression of a form of PKC-γ, phosphorylated on threonine 514 (pT514- PKC-γ), as well as the receptor for activated C kinase 1 (RACK1). The expression pattern of different PKC isoforms was altered in a complex and tissue-specific manner during chronic hyperglycemia. Notably, in the gray matter, PKC-α expression significantly decreased, while pT514-PKC-γ expression increased. However, PKC-βII, -γ, -δ, -ε, and RACK1 expressions did not change. Conversely, in glio-pial tissue, PKC-α and RACK1 were upregulated, whereas PKC-γ, pT514-PKCγ, and PKC-ε were downregulated. PKC-βII, and PKC-δ, were unchanged. These findings suggest that the PKC activity increase previously seen in the glio-pial tissue of diabetic rats may be due to the selective upregulation of PKC-α, and ultimately lead to the impairment of neurovascular coupling.

The effects of NG-nitro- l -arginine (NOLAG), an inhibitor of nitric oxide synthase (NOS), and of indomethacin, an inhibitor of cyclooxygenase, on the rise in cerebral blood flow (CBF) accompanying increasing levels of hypercapnia (paCO2 = 40–135mmHg) were studied in anesthetized rats. CBF was measured by intracarotid injection of 133Xe. Progressive increases in paCO2 of 10 mmHg, at intervals of about 8–10 minutes, were associated with gradual increases in CBF until a paCO2 level of 115 mmHg was reached. No further CBF changes (from the maximum value of 446 ± 70ml100g−1min−1) were seen with additional step increase in paCO2. Intracarotid infusion of 7.5 mg/kg NOLAG significantly attenuated the CO2-elicited CBF increase by about 45–65% at paCO2 values below 115 mmHg. Beyond this level, there was a lesser inhibition of about 27–35%. 30 mg/kg NOLAG had essentially the same effect as 7.5 mg/kg NOLAG. 50 mg/kg NOLAG, given intraperitoneally (i.p.) twice daily for 4 days, also caused an attenuated CBF response to CO2, but the inhibitory effect was significantly less than with acute NOLAG administration in the paCO2 range of 61–90 mmHg. Infusion of l -arginine, 1 g/kg/h, prevented the effect of 7.5 mg/kg NOLAG. Indomethacin, 10 mg/kg, i.v. produced a more dramatic attenuation of the response, to the extent that the steady rising curve of CBF as a function of paCO2 was almost completely abolished. With indomethacin, a moderate increase (50%) in CBF was seen at the lowest level of hypercapnia, but raising paCO2 above this level did not result in further increases in CBF. This effect could not be prevented by l -arginine. When combining 7.5 mg/kg NOLAG with 10 mg/kg indomethacin, the response to hypercapnia was totally blocked. The results suggest that NOLAG and indomethacin act through different mechanisms on the hypercapnic CBF response, and that indomethacin is the more powerful inhibitor.

In nondiabetic animals, estrogen has been shown to provide significant neuroprotection in focal and transient forebrain ischemia models. However, that neuroprotection may be diminished or lost in the diabetic. In this study, we compared the level of brain damage in intact, ovariectomized (OVX) and 17beta-estradiol (E(2))-treated OVX female rats rendered diabetic and chronically ( approximately 4 weeks) hyperglycemic via streptozotocin (STZ). Rats were subjected to 20 min of unilateral transient forebrain ischemia (reduction in cortical CBF to 20% of baseline). Neurologic function was analyzed daily and brain histopathology (in H&E-stained sections) was evaluated at 72 h of reperfusion. Supplemental histopathologic information was obtained from additional TUNEL-stained sections. When comparing neurologic outcome scores in the three groups, E(2)-treated OVX females displayed the highest degree of dysfunction and intact females the least (OVX rats not treated with E(2) were intermediate), with the difference between the intact and E(2)-treated groups being statistically significant. That same order was often observed with the regional histopathologic analyses of H&E-stained tissue. A significantly higher magnitude of neuronal loss in both OVX groups, when compared to intact females, was observed in the CA4 sector of the hippocampus and in the cortex. In addition, cell loss in the dorsal thalamus of the E(2)-treated group was significantly greater than in the intact females. Those results were generally corroborated by TUNEL-analysis, with 67% of the E(2)-treated, 33% of the control OVX, and only 17% of the intact females displaying TUNEL-positive cells in multiple regions. In conclusion, the present findings strongly suggest that the neuroprotective benefits of estrogen replacement therapy may be lost in the diabetic female rat.

The conclusion that cyclic 3′–5 guanosine monophosphate (cGMP) functions in a `permissive' manner in promoting cerebrovasodilation during hypercapnia was based on findings showing that the nitric oxide synthase (NOS) inhibitor-induced repression of the CO 2 response could be reversed upon addition of exogenous cGMP. We hypothesized that the action of cGMP revealed in those studies does not define its normal role in hypercapnic cerebral vasodilation, but rather is a unique function of the artificial situation of NOS inhibition coupled with cGMP repletion. Thus, although CO 2 reactivity may be the same in normal versus cGMP-repleted animals, the factors contributing to that response may differ. To test that possibility, the effects of calcium-dependent (K Ca ) or ATP-sensitive (K ATP ) potassium channel blockers on pial arteriolar CO 2 reactivity, in vivo, were evaluated in the presence and absence of NOS inhibition plus administration of a cGMP analogue. Pial arteriolar diameter changes in hypercapnia were measured in three principal groups of anesthetized rats: (I) K Ca channel-inhibited (via iberiotoxin); (II) K ATP channel-inhibited (via glibenclamide); and (III) controls. Group I and II rats were further divided into: (a) those treated with the neuronal NOS (nNOS) inhibitor, 7-nitroindazole (7-NI), followed by successive suffusions of the cGMP analogue, 8-bromo-cGMP (8Br-cGMP) and 8Br-cGMP+K-channel blocker; and (b) rats where 7-NI and 8Br-cGMP applications were omitted. Group III rats were divided into time and 8Br-cGMP controls. Hypercapnia (PCO 2 ≅60 mmHg, 3 min)-induced dilations were reduced by 70–80% following 7-NI and restored by 8Br-cGMP. That restoration was reversed by both K-channel blockers. In the absence of 7-NI and exogenous cGMP, CO 2 reactivity was unaffected by K-channel inhibition. These findings confirmed that nNOS-derived NO is critically important to the hypercapnic reactivity of cerebral arterioles, and that cGMP repletion, following NOS inhibition, could restore CO 2 reactivity. The observation that K Ca and K ATP channel blockade did not alter CO 2 reactivity under baseline conditions, but attenuated CO 2 reactivity only in the presence nNOS inhibition (and cGMP repletion), suggests that multiple, redundant, and interactive mechanisms participate in CO 2 -induced vasodilation. These results also imply that current strategies for revealing permissive actions of cGMP (or NO) may need to be re-evaluated.

In this study, we tested the hypothesis that nitric oxide (NO) and adenosine (ADO) are the principal mediators of severe hypoxia-induced vasodilation. In addition, we examined whether activation of N-methyl-D-aspartate (NMDA) receptors and/or perivascular nerves plays a role. A closed cranial window and intravital microscopy system was used to monitor diameter changes in pial arterioles (approximately 40 microns) in anesthetized rats. The relative contributions of ADO, NMDA, NO, and neuronal activation to hypoxic cerebrovasodilation were assessed using the blockers 8-sulfophenyltheophylline (8-SPT), MK-801, nitro-L-arginine methylester (L-NAME), and tetrodotoxin (TTX). Two experimental series were studied. In the first, we tested the effects of NOS inhibition, via topical L-NAME (1 mM), on moderate (PaO2 approximately 46 mmHg) then severe (PaO2 approximately 34 mmHg) hypoxia-induced dilation. To confirm that L-NAME was affecting specifically NO-dependent responses, we also examined, in each experiment, the vasodilatory responses to topical applications of NOS-dependent (adenosine diphosphate (ADP); acetylcholine (ACh)) and -independent (sodium nitroprusside (SNP)) agents, in the presence of L-NAME or, in controls, the presence of D-NAME or no added analogue. In the second series, topical suffusions of ADP, ADO, and NMDA were sequentially applied, followed by 5 min exposure to severe hypoxia (PaO2 approximately 32 mmHg). Following return to normoxia, a suffusion of either 8-SPT (10 microM), MK-801 (10 microM), TTX (1 microM), or 8-SPT+MK-801 was initiated (or, in controls, application of a drug-free suffusate was maintained), and the above sequence repeated. In control, TTX, and 8-SPT+MK-801 experiments, baseline conditions were then restored and hypercapnia (PaCO2 = 70-85 mmHg) was imposed. In the series 1 control groups, moderate and severe hypoxia elicited approximately 20% and 35-40% increases in diameter, respectively. L-NAME attenuated ADP- and ACh-induced dilations, did not alter the arteriolar responses to SNP or moderate hypoxia, but prevented further dilation upon imposition of severe hypoxia. This suggested that 45-50% of the severe hypoxia response was NO-dependent. In series 2, 8-SPT blocked the adenosine response and reduced severe hypoxia-induced dilation by 46%. MK-801 predictably blocked NMDA-induced relaxation and reduced the hypoxic response by 42%. When combined, 8-SPT and MK-801 affected hypoxic vasodilation additively. After TTX, the ADP and ADO responses were normal, but NMDA and hypoxia responses were completely blocked. Hypercapnia-induced dilation was unaffected by TTX or 8-SPT+MK-801. The results imply that severe hypoxia-induced release of NO and ADO, and the accompanying pial arteriolar dilation, are wholly dependent on the capacity to generate action potentials in perivascular nerves. The similarity of the L-NAME and MK-801 effects on hypoxic cerebrovasodilation suggests that the NO-dependency, to a large degree, derives from NMDA receptor activation.

Neurons synthesize NO, which may act as a retrograde messenger, involved in either potentiating or depressing neuronal excitability. NO may also play a role in the cerebral vasodilatory response to increased neuronal activity (i.e., seizures). In this study, two questions were asked: (1) is NO an endogenous anticonvulsant or proconvulsant substance? and (2) is the cerebral blood flow (CBF) increase accompanying bicuculline (BC)-induced seizures mediated by NO? The experiments were performed in 300-400-g Wistar rats anesthetized with 0.6% halothane and 70% N2O/30% O2. CBF was measured using the intracarotid 133Xe clearance method or laser-Doppler flowmetry. EEG activity was recorded. Chronic treatment (4 days) with nitro-L-arginine (L-NA), a potent NO synthase (NOS) inhibitor (400 mg/kg total), suppressed brain NOS by > 97% and prolonged seizure duration from 6 +/- 1 (saline-treated controls) to 12 +/- 2 min. In the L-NA-treated group, the CBF increase was sustained as long as seizure activity remained, indicating that CBF was still tightly coupled to seizure activity. Interestingly, the supposed inactive enantiomer of L-NA, D-NA, also showed an inhibition of brain NOS activity, ranging from 87 to 100%. The duration of seizures in this group (average 8 +/- 2 min) corresponded directly to the magnitude of reduction in NOS activity (r = 0.83, P < 0.05). Specifically, the D-NA results indicated that NOS inhibition had to exceed 95% before any effect on seizure duration could be seen.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional cerebral blood flow (rCBF) was measured in normoglycemic and chronically hypoglycemic rats. Chronic hypoglycemia was produced by continuously infusing insulin for 6-7 days. During chronic hypoglycemia (plasma glucose = 1.97 mumol/ml), rCBF increased in all regions except the cerebellum and hypothalamus. Blood flow increases present during chronic hypoglycemia were not as great as those previously measured during acute hypoglycemia. Therefore, adjustments in the regulation of rCBF occurred during chronic hypoglycemia compared to acute hypoglycemia.

In order to evaluate changes in extra- and intracellular pH in the brain during hypoglycemia rats were injected with insulin and pH changes evaluated when the EEG showed a slow-wave-polyspike pattern ('precoma'), or when EEG activity had ceased for 5, 15 or 30 min ('coma'). Extra- and intracellular acid-base changes were evaluated from pCO2 and HCO3-concentrations. In order to allow calculation of intracellular pH ( and HCO3- concentrations) changes in extracellular fluid volume were estimated by measurements of cortical tissue impedance. The main results were as follows. (1) At constant arterial pCO2 and CSF HCO3- concentration either rose (15 min of coma) or remained unchanged (all other groups). However, since the cerebrovenous (and tissue) pCO2 fell, all groups except one (30 min of coma) showed a significant increase in extracellular fluid pH. (2) During severe hypoglycemia, and especially when EEG activity ceased, cortical impedance increased markedly. Calculations with the help of the Rayleigh and Maxwell equations showed that the extracellular fluid volume was reduced to about 50% of control. (3) Intracellular pH increased significantly in precoma and in coma of 15 and 30 min duration. However, pH in the 5 min coma group was significantly lower (but no different from control). (4) In general, the increase in intracellular pH is consistent with previous findings that hypoglycemia is associated with oxidation of endogenous acid metabolites. However, the data suggest that in the initial period of coma acids accumulate by some unidentified mechanism.

The respiratory function of cerebral mitochondria harvested from genetically diabetic (BB/W) and streptozotocin-diabetic rats deprived of insulin for 3–4 weeks was found to be unchanged from control values. Furthermore, insulin-deprived BB/W rats subjected to 30 min of insulin-induced hypoglycemic coma demonstrated a normal mitochondrial respiration following a 60 min period of glucose restitution, a finding consistent with earlier results in non-diabetic rats. However, in rats exposed to 1 week of moderate hypoglycemia (plasma glucose = 3.0 μmol·ml−1), both state 3 respiration and the respiratory control ratio (RCR) were reduced from control. In fact, when the chronic hypoglycemia was imposed following a 3–4 week period of diabetic hyperglycemia, the state 3 rate and RCR were found to be reduced to a greater degree than in chronically hypoglycemic, non-diabetic, previously normoglycemic rats. Finally, when 1 week of moderate hypoglycemia preceded a 30 min period of insulin-induced hypoglycemic coma, a disturbed pattern of mitochondrial respiration (i.e. increased state 4, decreased RCR) was found at 60 min of recovery following coma. These results indicate that chronic increases in glucose (and insulin deprivation) have no effect on cerebral mitochondrial respiratory function, whereas prolonged, albeit moderate, reductions in cerebral glucose supply result in perturbations in mitochondrial respiration. These results demonstrate the importance of an adequate glucose supply for normal mitochondrial activity.