We measured quantitative cortical mantle cerebral blood flow (CBF) by stable xenon computed tomography (CT) within the first 12 h after severe traumatic brain injury (TBI) to determine whether neurologic outcome can be predicted by CBF stratification early after injury. Stable xenon CT was used for quantitative measurement of CBF (mL/100 g/min) in 22 cortical mantle regions stratified as follows: low (0-8), intermediate (9-30), normal (31-70), and hyperemic (>70) in 120 patients suffering severe (Glasgow Coma Scale [GCS] score ≤8) TBI. For each of these CBF strata, percentages of total cortical mantle volume were calculated. Outcomes were assessed by Glasgow Outcome Scale (GOS) score at discharge (DC), and 1, 3, and 6 months after discharge. Quantitative cortical mantle CBF differentiated GOS 1 and GOS 2 (dead or vegetative state) from GOS 3-5 (severely disabled to good recovery; p<0.001). Receiver operating characteristic (ROC) curve analysis for percent total normal plus hyperemic flow volume (TNHV) predicting GOS 3-5 outcome at 6 months for CBF measured <6 and <12 h after injury showed ROC area under the curve (AUC) cut-scores of 0.92 and 0.77, respectively. In multivariate analysis, percent TNHV is an independent predictor of GOS 3-5, with an odds ratio of 1.460 per 10 percentage point increase, as is initial GCS score (OR=1.090). The binary version of the Marshall CT score was an independent predictor of 6-month outcome, whereas age was not. These results suggest that quantitative cerebral cortical CBF measured within the first 6 and 12 h after TBI predicts 6-month outcome, which may be useful in guiding patient care and identifying patients for randomized clinical trials. A larger multicenter randomized clinical trial is indicated.

Hypertension, diabetes, obesity, and dyslipidemia are risk factors that characterize metabolic syndrome (MetS), which increases the risk for stroke by 40%. In a preliminary study, our aim was to evaluate cerebrovascular reactivity and oxygen metabolism in subjects free of vascular disease but with one or more of these risk factors. Volunteers (n=15) 59±15 (mean±SD)years of age clear of cerebrovascular disease by magnetic resonance angiography but with one or more risk factors were studied by quantitative positron emission tomography for measure ment of cerebral blood flow, oxygen consumption, oxygen extraction fraction (OEF), and acetazolamide cerebrovascular reactivity. Eight of ten subjects with MetS risk factors had OEF >50%. None of the five without risk factors had OEF >50%. The presence of MetS risk factors was highly correlated with OEF >50% by Fisher's exact test (p<0.007). The increase in OEF was significantly (P<0.001) correlated with cerebral metabolic rate for oxygen. Increased OEF was not associated with compromised acetazolamide cerebrovascular reactivity. Subjects with one or more MetS risk factors are characterized by increased cerebral oxygen consumption and ischemic stress, which may be related to increased risk of cerebrovascular disease and stroke.

BACKGROUND AND PURPOSE: The Carotid Occlusion Surgery Study (COSS) was an improvement over the Extracranial-Intracranial Bypass Study, which did not utilize physiological selection. To assess possible reasons for early closure of the COSS trial, we reviewed COSS methods used to identify high-risk patients and compared results with separate quantitative data. METHODS: Increased oxygen extraction fraction (OEF) by positron emission tomography is a gold standard for ischemia, but the specific thresholds and equivalency of the semiquantitative OEF ratio utilized in COSS and quantitative OEF are at issue. RESULTS: The semiquantitative hemispheric OEF ratio used in COSS did not identify the same group of patients as did quantitative OEF using a threshold of 50%. CONCLUSIONS: The failure of COSS is likely caused by a failure of the semiquantitative, hemispheric OEF ratio method rather than by the selection for bypass based on hemodynamic compromise.

The basic premise of neuroprotection in acute stroke is the presence of salvageable tissue, but the spatiotemporal volume profiles of the penumbra and infarction remain poorly defined in preclinical animal models of acute stroke used to evaluate therapies for clinical application. Our aim was to define these profiles using magnetic resonance imaging (MRI) quantitative cerebral blood flow (CBF) and apparent diffusion coefficient (ADC) for dual-parameter voxel analysis in the rat suture permanent middle cerebral artery occlusion (pMCAO) model. Eleven male Sprague Dawley rats were subjected to pMCAO with MRI measurements of quantitative CBF and ADC at baseline, over the first 4 h (n=9) and at 7, 14, and 21 days (n=4). Voxel analysis of CBF and ADC was used to characterize brain tissue ischemic transitions. Penumbra, core, and hyperemic infarction volumes were significantly elevated (P<0.05) and unchanged over the first 4 h of pMCAO while the total lesion volume progressively rose. At 7, 14, and 21 days, tissue compartment transitions reflected infarction, tissue cavitation, and selective ischemic neuronal necrosis. Anatomical distribution of penumbra and core revealed marked heterogeneity with penumbra scattered within core and penumbra persisting even after 4 h of permanent MCAO.

The cytochrome P450 (CYP) isoforms that catalyze the oxidation metabolism of 6-methoxy-2-napthylacetic acid (6-MNA), an active metabolite of nabumetone, were studied in rats and humans. Using an extractive reversed-phase HPLC assay with fluorescence detection, monophasic Michaelis-Menten kinetics was obtained for the formation of 6-hydroxy-2-naphthylacetic acid (6-HNA) in liver microsomes of rats and humans, and kinetic analysis showed that the K(m) and V(max) values for the formation of 6-HNA in humans and rats were 640.0 ± 30.9 and 722.9 ± 111.7 µM, and 1167.5 ± 33.0 and 1312.7 ± 73.8 pmol min⁻¹ mg protein⁻¹, respectively. The CYPs responsible for metabolism of 6-MNA in liver microsomes of rats and humans were identified using correlation study, recombinant CYP supersomes, and specific CYP inhibitors and antibodies. Recombinant human CYP2C9 exhibited appreciable catalytic activity with respect to 6-HNA formation from 6-MNA. Among 14 recombinant rat CYPs examined, CYP2C6, CYP2C11 and CYP1A2 were involved in the metabolism of 6-MNA. Sulfaphenazole (a selective inhibitor of CYP2C9) inhibited the formation of 6-HNA in pooled human microsomes by 89%, but failed to inhibit this reaction in rat liver microsomes. The treatment of pooled human liver microsomes with an antibody against CYP2C9 inhibited the formation of 6-HNA by about 80%. The antibody against CYP2C11 suppressed the activity by 20 to 30% in rat microsomes, whereas that of CYP1A2 microsomes did not show drastic inhibition. These findings suggest that CYP2C9 has the highest catalytic activity of 6-MNA metabolism in humans. In contrast, metabolism of 6-MNA is suggested to be mediated mainly by CYP2C6 and CYP2C11 in rats.

Extracellular phosphate (Pi) is known to play a key role in promoting osteoblastic differentiation by altering gene expression and cellular function. Importantly, it may be possible to use this knowledge as a means to deliver Pi to local sites to regenerate mineralized tissues associated with the oral cavity. Therefore, we determined the ability of Pi to regulate differentiation of pulp cells toward an odontoblast phenotype and further determined if this was in part due to an increase in the expression of bone morphogenetic protein (BMP)-2, a crucial regulator of mineralization. Results showed that Pi increased BMP-2 expression at both mRNA and protein level and BMP-2 promoter activity. Signaling inhibitors revealed that increased BMP-2 expression was dependent on cAMP/protein kinase A but not the protein kinase C signaling pathway. Treatment with 8-Br-cAMP, a cell-permeable analog of cAMP, enhanced Pi-mediated BMP-2 expression, but treatment with 8-Br-cAMP alone did not increase BMP-2, suggesting that cAMP is indispensable but not sufficient for Pi-mediated BMP-2 expression. Pi activated ERK1/2, and treatment with PD98059, an ERK1/2 inhibitor, suppressed Pi-mediated BMP-2 increase, indicating a requirement for activation of ERK1/2. ERK1/2 pathway may operate independently of cAMP-dependent signaling because MDL12,330A, an adenylate cyclase inhibitor, did not inhibit phosphorylation of ERK1/2 in response to Pi. Pulp cells expressed the sodium-dependent Pi transporter (NaPi) III type, but not NaPi-I type or NaPi-II type. Pi-mediated BMP-2 increase was inhibited in the presence of phosphonoformic acid, an inhibitor not only of NaPi transport but also of crystal nucleation. Furthermore, a similar inhibition was observed in the presence of pyrophosphate, a mineralization inhibitor. These findings demonstrate, for the first time, that Pi regulates BMP-2 expression via cAMP/protein kinase A and ERK1/2 pathways in human dental pulp cells.

To manage patients with high intracranial pressure (ICP), clinicians need to know the critical cerebral perfusion pressure (CPP) required to maintain cerebral blood flow (CBF). Historically, the critical CPP obtained by decreasing mean arterial pressure (MAP) to lower CPP was 60 mm Hg, which fell to 30 mm Hg when CPP was reduced by increasing ICP. We examined whether this decrease in critical CPP was due to a pathological shift from capillary (CAP) to high-velocity microvessel flow or thoroughfare channel (TFC) shunt flow. Cortical microvessel red blood cell velocity and NADH fluorescence were measured by in vivo two-photon laser scanning microscopy in rats at CPP of 70, 50, and 30 mm Hg by increasing ICP or decreasing MAP. Water content was measured by wet/dry weight, and cortical perfusion by laser Doppler flux. Reduction of CPP by raising ICP increased TFC shunt flow from 30.4±2.3% to 51.2±5.2%(mean±SEM, p<0.001), NADH increased by 20.3±6.8% and 58.1±8.2%(p<0.01), and brain water content from 72.9±0.47% to 77.8±2.42%(p<0.01). Decreasing CPP by MAP decreased TFC shunt flow with a smaller rise in NADH and no edema. Doppler flux decreased less with increasing ICP than decreasing MAP. The decrease seen in the critical CPP with increased ICP is likely due to a redistribution of microvascular flow from capillary to microvascular shunt flow or TFC shunt flow, resulting in a pathologically elevated CBF associated with tissue hypoxia and brain edema, characteristic of non-nutritive shunt flow.

Cementoblasts, tooth root lining cells, are responsible for laying down cementum on the root surface, a process that is indispensable for establishing a functional periodontal ligament. Cementoblasts share phenotypical features with osteoblasts. Elevated levels of extracellular Ca(2+) have been implicated in osteogenesis by stimulating the proliferation and differentiation of osteoblasts; however, the role of extracellular Ca(2+) signaling in cementogenesis has not been examined. Using RT-PCR, we found that elevated levels of extracellular Ca(2+) increase fibroblast growth factor (FGF)-2 gene expression with a peak at 6h. Pretreatment with a protein kinase A (PKA) inhibitor, H89, or an adenylate cyclase inhibitor, MDL-12,330A, inhibited Ca(2+)-stimulated Fgf-2 expression. In contrast, pretreatment with the protein kinase C (PKC) inhibitor GF-109203X or the phospholipase C (PLC) inhibitor U73122 did not affect the expression of Fgf-2 transcripts, suggesting that the increase in Fgf-2 expression was dependent on the PKA but not the PLC/PKC signaling pathway. Treatment with an activator of adenylate cyclase, forskolin, or a cell-permeable analog of cAMP, 8-Br-cAMP, enhanced Ca(2+)-stimulated Fgf-2 expression, but a single treatment with forskolin or 8-Br-cAMP did not, suggesting that cAMP generation is indispensable but not sufficient for Ca(2+)-stimulated FGF2 expression. Next, we examined the cation specificity of the putative receptor and showed that treatment with trivalent/divalent inorganic ions, Ca(2+), Gd(3+), Sr(2+), or Al(3+), caused a dose-dependent increase in Fgf-2 mRNA levels in a cAMP-dependent fashion, whereas Mg(2+) and the organic ions neomycin and spermine had no effect on Fgf-2 gene expression levels. These findings suggest that an extracellular Ca(2+)-sensing mechanism is present in cementoblasts and its activation leads to FGF-2 stimulation in a cAMP/PKA dependent fashion. Understanding the pathway regulating key genes involved in modulating the regeneration of oral tissues will assist in designing regenerative therapies based on reliable biological principles.

Signaling by the Wnt plays a central role in many processes during embryonic development and adult homeostasis. At least 19 types of Wnts, several families of secreted antagonists and multiple receptors have been identified. Two distinct Wnt signaling pathways, the canonical pathway and the noncanonical pathway have been described. Functional studies and experimental analysis of relevant animal models confirmed the effects of Wnt on regulation of developing mineralized tissue formation and adult homeostasis. In osteoblasts, the canonical Wnt pathway modulates differentiation, proliferation and mineralization, while it blocks apoptosis and osteoclastogenesis by increasing osteoprotegerin. Functional crosstalk between Wnt and bone morphogenetic protein signaling during osteoblastic differentiation has been reported. Recently, non-canonical Wnt signaling was shown to play a role in bone formation. The Wnt signaling pathway also plays an important role not only in tooth formation but also in differentiation and proliferation of cementoblasts and odontoblasts in the tooth. This present review provides an overview of progress in elucidating the role of Wnt signaling pathways in bone and tooth and the resulting possibilities for therapeutic potential.

Dental pulp cells, which have been shown to share phenotypical features with osteoblasts, are capable of differentiating into odontoblast-like cells and generating a dentin-like mineral structure. Elevated extracellular Ca(2+)(Ca(2+)(o)) has been implicated in osteogenesis by stimulating the proliferation and differentiation of osteoblasts; however, the role of Ca(2+)(o) signaling in odontogenesis remains unclear. We found that elevated Ca(2+)(o) increases bone morphogenetic protein (BMP)-2 gene expression in human dental pulp cells. The increase was modulated not only at a transcriptional level but also at a post-transcriptional level, because treatment with Ca(2+) increased the stability of BMP-2 mRNA in the presence of actinomycin D, an inhibitor of transcription. A similar increase in BMP-2 mRNA level was observed in other human mesenchymal cells from oral tissue; periodontal ligament cells and gingival fibroblasts. However, the latter cells exhibited considerably lower expression of BMP-2 mRNA compared with dental pulp cells and periodontal ligament cells. The BMP-2 increase was markedly inhibited by pretreatment with an extracellular signal-regulated kinase (ERK) inhibitor, PD98059, and partially inhibited by the L-type Ca(2+) channels inhibitor, nifedipine. However, pretreatment with nifedipine had no effect on ERK1/2 phosphorylation triggered by Ca(2+), suggesting that the Ca(2+) influx from Ca(2+) channels may operate independently of ERK signaling. Dental pulp cells do not express the transcript of Ca(2+)-sensing receptors (CaSR) and only respond slightly to other cations such as Sr(2+) and spermine, suggesting that dental pulp cells respond to Ca(2+)(o) to increase BMP-2 mRNA expression in a manner different from CaSR and rather specific for Ca(2+)(o) among cations.