Vascular imprints of neuronal activity: Relationships between the dynamics of cortical blood flow, oxygenation, and volume changes following sensory stimulation

Modern functional neuroimaging methods, such as positron-emission tomography (PET), optical imaging of intrinsic signals, and functional MRI (fMRI) utilize activity-dependent hemodynamic changes to obtain indirect maps of the evoked electrical activity in the brain. Whereas PET and flow-sensitive MRI map cerebral blood flow (CBF) changes, optical imaging and blood oxygenation level-dependent MRI map areas with changes in the concentration of deoxygenated hemoglobin (HbR). However, the relationship between CBF and HbR during functional activation has never been tested experimentally. Therefore, we investigated this relationship by using imaging spectroscopy and laser-Doppler flowmetry techniques, simultaneously, in the visual cortex of anesthetized cats during sensory stimulation. We found that the earliest microcirculatory change was indeed an increase in HbR, whereas the CBF increase lagged by more than a second after the increase in HbR. The increased HbR was accompanied by a simultaneous increase in total hemoglobin concentration (Hbt), presumably reflecting an early blood volume increase. We found that the CBF changes lagged after Hbt changes by 1 to 2 sec throughout the response. These results support the notion of active neurovascular regulation of blood volume in the capillary bed and the existence of a delayed, passive process of capillary filling.

Blood pressure reduction and diabetes insipidus in transgenic rats deficient in brain angiotensinogen

Angiotensin produced systemically or locally in tissues such as the brain plays an important role in the regulation of blood pressure and in the development of hypertension. We have established transgenic rats [TGR(ASrAOGEN)] expressing an antisense RNA against angiotensinogen mRNA specifically in the brain. In these animals, the brain angiotensinogen level is reduced by more than 90% and the drinking response to intracerebroventricular renin infusions is decreased markedly compared with control rats. Blood pressure of transgenic rats is lowered by 8 mmHg (1 mmHg = 133 Pa) compared with control rats. Crossbreeding of TGR(ASrAOGEN) with a hypertensive transgenic rat strain exhibiting elevated angiotensin II levels in tissues results in a marked attenuation of the hypertensive phenotype. Moreover, TGR(ASrAOGEN) exhibit a diabetes insipidus-like syndrome producing an increased amount of urine with decreased osmolarity. The observed reduction in plasma vasopressin by 35% may mediate these phenotypes of TGR(ASrAOGEN). This new animal model presenting long-term and tissue-specific down-regulation of angiotensinogen corroborates the functional significance of local angiotensin production in the brain for the central regulation of blood pressure and for the pathogenesis of hypertension.

Detection and characterization of carcinoma cells in the blood

A highly sensitive assay combining immunomagnetic enrichment with multiparameter flow cytometric and immunocytochemical analysis has been developed to detect, enumerate, and characterize carcinoma cells in the blood. The assay can detect one epithelial cell or less in 1 ml of blood. Peripheral blood (10–20 ml) from 30 patients with carcinoma of the breast, from 3 patients with prostate cancer, and from 13 controls was examined by flow cytometry for the presence of circulating epithelial cells defined as nucleic acid+, CD45−, and cytokeratin+. Highly significant differences in the number of circulating epithelial cells were found between normal controls and patients with cancer including 17 with organ-confined disease. To determine whether the circulating epithelial cells in the cancer patients were neoplastic cells, cytospin preparations were made after immunomagnetic enrichment and were analyzed. Epithelial cells from patients with breast cancer generally stained with mAbs against cytokeratin and 3 of 5 for mucin-1. In contrast, no cells that stained for these antigens were observed in the blood from normal controls. The morphology of the stained cells was consistent with that of neoplastic cells. Of 8 patients with breast cancer followed for 1–10 months, there was a good correlation between changes in the level of tumor cells in the blood with both treatment with chemotherapy and clinical status. The present assay may be helpful in early detection, in monitoring disease, and in prognostication.

Dynamic mapping at the laminar level of odor-elicited responses in rat olfactory bulb by functional MRI

We have applied functional MRI (fMRI) based on blood oxygenation level-dependent (BOLD) image-contrast to map odor-elicited olfactory responses at the laminar level in the rat olfactory bulb (OB) elicited by iso-amyl acetate (10−2 dilution of saturated vapor) with spatial and temporal resolutions of 220×220×1,000 μm and 36 s. The laminar structure of the OB was clearly depicted by high-resolution in vivo anatomical MRI with spatial resolution of 110×110×1,000 μm. In repeated BOLD fMRI measurements, highly significant (P < 0.001) foci were located in the outer layers of both OBs. The occurrence of focal OB activity within a domain at the level of individual glomeruli or groups of glomeruli was corroborated on an intra- and inter-animal basis under anesthetized conditions with this noninvasive method. The dynamic studies demonstrated that the odor-elicited BOLD activations were highly reproducible on a time scale of minutes, whereas over tens of minutes the activations sometimes varied slowly. We found large BOLD signal (ΔS/S = 10–30%) arising from the olfactory nerve layer, which is devoid of synapses and composed of unmyelinated fibers and glial cells. Our results support previous studies with other methods showing that odors elicit activity within glomerular layer domains in the mammalian OB, and extend the analysis to shorter time periods at the level of individual glomeruli or groups of glomeruli. With further improvement, BOLD fMRI should be ideal for systematic analysis of the functional significance of individual glomeruli in olfactory information encoding and of spatiotemporal processing within the olfactory system.

Two hemoglobin genes in Arabidopsis thaliana: The evolutionary origins of leghemoglobins

We cloned two hemoglobin genes from Arabidopsis thaliana. One gene, AHB1, is related in sequence to the family of nonsymbiotic hemoglobin genes previously identified in a number of plant species (class 1). The second hemoglobin gene, AHB2, represents a class of nonsymbiotic hemoglobin (class 2) related in sequence to the symbiotic hemoglobin genes of legumes and Casuarina. The properties of these two hemoglobins suggest that the two families of nonsymbiotic hemoglobins may differ in function from each other and from the symbiotic hemoglobins. AHB1 is induced, in both roots and rosette leaves, by low oxygen levels. Recombinant AHB1 has an oxygen affinity so high as to make it unlikely to function as an oxygen transporter. AHB2 is expressed at a low level in rosette leaves and is low temperature-inducible. AHB2 protein has a lower affinity for oxygen than AHB1 but is similar to AHB1 in having an unusually low, pH-sensitive oxygen off-rate.

Transgenic knockout mice exclusively expressing human hemoglobin S after transfer of a 240-kb βs-globin yeast artificial chromosome: A mouse model of sickle cell anemia

Sickle cell anemia (SCA) and thalassemia are among the most common genetic diseases worldwide. Current approaches to the development of murine models of SCA involve the elimination of functional murine α- and β-globin genes and substitution with human α and βs transgenes. Recently, two groups have produced mice that exclusively express human HbS. The transgenic lines used in these studies were produced by coinjection of human α-, γ-, and β-globin constructs. Thus, all of the transgenes are integrated at a single chromosomal site. Studies in transgenic mice have demonstrated that the normal gene order and spatial organization of the members of the human β-globin gene family are required for appropriate developmental and stage-restricted expression of the genes. As the cis-acting sequences that participate in activation and silencing of the γ- and β-globin genes are not fully defined, murine models that preserve the normal structure of the locus are likely to have significant advantages for validating future therapies for SCA. To produce a model of SCA that recapitulates not only the phenotype, but also the genotype of patients with SCA, we have generated mice that exclusively express HbS after transfer of a 240-kb βs yeast artificial chromosome. These mice have hemolytic anemia, 10% irreversibly sickled cells in their peripheral blood, reticulocytosis, and other phenotypic features of SCA.

U94 of human herpesvirus 6 is expressed in latently infected peripheral blood mononuclear cells and blocks viral gene expression in transformed lymphocytes in culture

Human herpesvirus 6 (HHV-6) like other herpesviruses, expresses sequentially immediate early (IE), early, and late genes during lytic infection. Evidence of ability to establish latent infection has not been available, but by analogy with other herpesviruses it could be expected that IE genes that regulate and transactivate late genes would not be expressed. We report that peripheral blood mononuclear cells of healthy individuals infected with HHV-6 express the U94 gene, transcribed under IE conditions. Transcription of other IE genes (U16/17, U39, U42, U81, U89/90, U91) was not detected. To verify that U94 may play a role in the maintenance of the latent state, we derived lymphoid cell lines that stably expressed U94. HHV-6 was able to infect these cells, but viral replication was restricted. No cytopathic effect developed. Furthermore, viral transcripts were present in the first days postinfection and declined thereafter. A similar decline in the level of intracellular viral DNA also was observed. These findings are consistent with the hypothesis that the U94 gene product of HHV-6 regulates viral gene expression and enables the establishment and/or maintenance of latent infection in lymphoid cells.

Long-term expression of γ-globin mRNA in mouse erythrocytes from retrovirus vectors containing the human γ-globin gene fused to the ankyrin-1 promoter

Gene therapy for patients with hemoglobin disorders has been hampered by the inability of retrovirus vectors to transfer globin genes and their cis-acting regulatory sequences into hematopoietic stem cells without rearrangement. In addition, the expression from intact globin gene vectors has been variable in red blood cells due to position effects and retrovirus silencing. We hypothesized that by substituting the globin gene promoter for the promoter of another gene expressed in red blood cells, we could generate stable retrovirus vectors that would express globin at sufficient levels to treat hemoglobinopathies. Recently, we have shown that the human ankyrin (Ank) gene promoter directs position-independent, copy number-dependent expression of a linked γ-globin gene in transgenic mice. We inserted the Ank/Aγ-globin gene into retrovirus vectors that could transfer one or two copies of the Ank/Aγ-globin gene to target cells. Both vectors were stable, transferring only intact proviral sequences into primary mouse hematopoietic stem cells. Expression of Ank/Aγ-globin mRNA in mature red blood cells was 3% (single copy) and 8% (double copy) of the level of mouse α-globin mRNA. We conclude that these novel retrovirus vectors may be valuable for treating a variety of red cell disorders by gene replacement therapy including severe β-thalassemia if the level of expression can be further increased.

Relative role of heme nitrosylation and β-cysteine 93 nitrosation in the transport and metabolism of nitric oxide by hemoglobin in the human circulation

To quantify the reactions of nitric oxide (NO) with hemoglobin under physiological conditions and to test models of NO transport on hemoglobin, we have developed an assay to measure NO–hemoglobin reaction products in normal volunteers, under basal conditions and during NO inhalation. NO inhalation markedly raised total nitrosylated hemoglobin levels, with a significant arterial–venous gradient, supporting a role for hemoglobin in the transport and delivery of NO. The predominant species accounting for this arterial–venous gradient is nitrosyl(heme)hemoglobin. NO breathing increases S-nitrosation of hemoglobin β-chain cysteine 93, however only to a fraction of the level of nitrosyl(heme)hemoglobin and without a detectable arterial–venous gradient. A strong correlation between methemoglobin and plasma nitrate formation was observed, suggesting that NO metabolism is a primary physiological cause of hemoglobin oxidation. Our results demonstrate that NO–heme reaction pathways predominate in vivo, NO binding to heme groups is a rapidly reversible process, and S-nitrosohemoglobin formation is probably not a primary transport mechanism for NO but may facilitate NO release from heme.

Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex

Cortical blood flow at the level of individual capillaries and the coupling of neuronal activity to flow in capillaries are fundamental aspects of homeostasis in the normal and the diseased brain. To probe the dynamics of blood flow at this level, we used two-photon laser scanning microscopy to image the motion of red blood cells (RBCs) in individual capillaries that lie as far as 600 μm below the pia mater of primary somatosensory cortex in rat; this depth encompassed the cortical layers with the highest density of neurons and capillaries. We observed that the flow was quite variable and exhibited temporal fluctuations around 0.1 Hz, as well as prolonged stalls and occasional reversals of direction. On average, the speed and flux (cells per unit time) of RBCs covaried linearly at low values of flux, with a linear density of ≈70 cells per mm, followed by a tendency for the speed to plateau at high values of flux. Thus, both the average velocity and density of RBCs are greater at high values of flux than at low values. Time-locked changes in flow, localized to the appropriate anatomical region of somatosensory cortex, were observed in response to stimulation of either multiple vibrissae or the hindlimb. Although we were able to detect stimulus-induced changes in the flux and speed of RBCs in some single trials, the amplitude of the stimulus-evoked changes in flow were largely masked by basal fluctuations. On average, the flux and the speed of RBCs increased transiently on stimulation, although the linear density of RBCs decreased slightly. These findings are consistent with a stimulus-induced decrease in capillary resistance to flow.

Correlation of breath ammonia with blood urea nitrogen and creatinine during hemodialysis

We have spectroscopically determined breath ammonia levels in seven patients with end-stage renal disease while they were undergoing hemodialysis at the University of California, Los Angeles, dialysis center. We correlated these measurements against simultaneously taken blood samples that were analyzed for blood urea nitrogen (BUN) and creatinine, which are the accepted standards indicating the level of nitrogenous waste loading in a patient's bloodstream. Initial levels of breath ammonia, i.e., at the beginning of dialysis, are between 1,500 ppb and 2,000 ppb (parts per billion). These levels drop very sharply in the first 15–30 min as the dialysis proceeds. We found the reduction in breath ammonia concentration to be relatively slow from this point on to the end of dialysis treatment, at which point the levels tapered off at 150 to 200 ppb. For each breath ammonia measurement, taken at 15–30 min intervals during the dialysis, we also sampled the patient's blood for BUN and creatinine. The breath ammonia data were available in real time, whereas the BUN and creatinine data were available generally 24 h later from the laboratory. We found a good correlation between breath ammonia concentration and BUN and creatinine. For one of the patients, the correlation gave an R2 of 0.95 for breath ammonia and BUN correlation and an R2 of 0.83 for breath ammonia and creatinine correlation. These preliminary data indicate the possibility of using the real-time breath ammonia measurements for determining efficacy and endpoint of hemodialysis.

A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood

The integrity of cell membranes is maintained by a balance between the amount of cholesterol and the amounts of unsaturated and saturated fatty acids in phospholipids. This balance is maintained by membrane-bound transcription factors called sterol regulatory element-binding proteins (SREBPs) that activate genes encoding enzymes of cholesterol and fatty acid biosynthesis. To enhance transcription, the active NH2-terminal domains of SREBPs are released from endoplasmic reticulum membranes by two sequential cleavages. The first is catalyzed by Site-1 protease (S1P), a membrane-bound subtilisin-related serine protease that cleaves the hydrophilic loop of SREBP that projects into the endoplasmic reticulum lumen. The second cleavage, at Site-2, requires the action of S2P, a hydrophobic protein that appears to be a zinc metalloprotease. This cleavage is unusual because it occurs within a membrane-spanning domain of SREBP. Sterols block SREBP processing by inhibiting S1P. This response is mediated by SREBP cleavage-activating protein (SCAP), a regulatory protein that activates S1P and also serves as a sterol sensor, losing its activity when sterols overaccumulate in cells. These regulated proteolytic cleavage reactions are ultimately responsible for controlling the level of cholesterol in membranes, cells, and blood.

Blood flow and oxygen delivery to human brain during functional activity: Theoretical modeling and experimental data

Coupling of cerebral blood flow (CBF) and cerebral metabolic rate for oxygen (CMRO2) in physiologically activated brain states remains the subject of debates. Recently it was suggested that CBF is tightly coupled to oxidative metabolism in a nonlinear fashion. As part of this hypothesis, mathematical models of oxygen delivery to the brain have been described in which disproportionately large increases in CBF are necessary to sustain even small increases in CMRO2 during activation. We have explored the coupling of CBF and oxygen delivery by using two complementary methods. First, a more complex mathematical model was tested that differs from those recently described in that no assumptions were made regarding tissue oxygen level. Second, [15O] water CBF positron emission tomography (PET) studies in nine healthy subjects were conducted during states of visual activation and hypoxia to examine the relationship of CBF and oxygen delivery. In contrast to previous reports, our model showed adequate tissue levels of oxygen could be maintained without the need for increased CBF or oxygen delivery. Similarly, the PET studies demonstrated that the regional increase in CBF during visual activation was not affected by hypoxia. These findings strongly indicate that the increase in CBF associated with physiological activation is regulated by factors other than local requirements in oxygen.

Improved retroviral gene transfer into murine and Rhesus peripheral blood or bone marrow repopulating cells primed in vivo with stem cell factor and granulocyte colony-stimulating factor.

In previous studies we showed that 5 days of treatment with granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) mobilized murine repopulating cells to the peripheral blood (PB) and that these cells could be efficiently transduced with retroviral vectors. We also found that, 7-14 days after cytokine treatment, the repopulating ability of murine bone marrow (BM) increased 10-fold. In this study we examined the efficiency of gene transfer into cytokine-primed murine BM cells and extended our observations to a nonhuman primate autologous transplantation model. G-CSF/SCF-primed murine BM cells collected 7-14 days after cytokine treatment were equivalent to post-5-fluorouracil BM or G-CSF/SCF-mobilized PB cells as targets for retroviral gene transfer. In nonhuman primates, CD34-enriched PB cells collected after 5 days of G-CSF/SCF treatment and CD34-enriched BM cells collected 14 days later were superior targets for retroviral gene transfer. When a clinically approved supernatant infection protocol with low-titer vector preparations was used, monkeys had up to 5% of circulating cells containing the vector for up to a year after transplantation. This relatively high level of gene transfer was confirmed by Southern blot analysis. Engraftment after transplantation using primed BM cells was more rapid than that using steady-state bone marrow, and the fraction of BM cells saving the most primitive CD34+/CD38- or CD34+/CD38dim phenotype increased 3-fold. We conclude that cytokine priming with G-CSF/SCF may allow collection of increased numbers of primitive cells from both the PB and BM that have improved susceptibility to retroviral transduction, with many potential applications in hematopoietic stem cell-directed gene therapy.

Reduction in the level of Gal(alpha1,3)Gal in transgenic mice and pigs by the expression of an alpha(1,2)fucosyltransferase.

Hyperacute rejection of a porcine organ by higher primates is initiated by the binding of xenoreactive natural antibodies of the recipient to blood vessels in the graft leading to complement activation. The majority of these antibodies recognize the carbohydrate structure Gal(alphal,3)Gal (gal epitope) present on cells of pigs. It is possible that the removal or lowering of the number of gal epitopes on the graft endothelium could prevent hyperacute rejection. The Gal(alpha1,3) Gal structure is formed by the enzyme Galbeta1,4GlcNAc3-alpha-D-galactosyltransferase [alpha(1,3)GT; EC 2.4.1.51], which transfers a galactose molecule to terminal N-acetyllactosamine (N-lac) present on various glycoproteins and glycolipids. The N-lac structure might be utilized as an acceptor by other glycosyltransferases such as Galbeta1,4GlcNAc 6-alpha-D-sialyltransferase [alpha(2,6)ST], Galbeta1,4GlcNAc 3-alpha-D-Sialyltransferase [alpha(2,3)ST], or Galbeta 2-alpha-L-fucosyltransferase [alpha(1,2)FT; EC 2.4.1.691, etc. In this report we describe the competition between alpha(1,2)FT and alpha(1,3)GT in cells in culture and the generation of transgenic mice and transgenic pigs that express alpha(1,2)Fr leading to synthesis of Fucalpha,2Galbeta- (H antigen) and a concomitant decrease in the level of Gal(alpha1,3)Gal. As predicted, this resulted in reduced binding of xenoreactive natural antibodies to endothelial cells of transgenic mice and protection from complement mediated lysis.