Brain temperature by Biosensor Imaging of Redundant Deviation in Shifts (BIRDS): comparison between TmDOTP5−and TmDOTMA−

Chemical shifts of complexes between paramagnetic lanthanide ions and macrocyclic chelates are sensitive to physiological variations (of temperature and/or pH). Here we demonstrate utility of a complex between thulium ion (Tm³⁺) and the macrocyclic chelate 1,4,7,10‐tetramethyl 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetate (or DOTMA^4?) for absolute temperature mapping in rat brain. The feasibility of TmDOTMA^? is compared with that of another Tm³⁺‐containing biosensor which is based on the macrocyclic chelate 1,4,7,10‐tetraazacyclododecane‐ 1,4,7,10‐tetrakis(methylene phosphonate) (or DOTP^8?). In general, the in vitro and in vivo results suggest that Biosensor Imaging of Redundant Deviation in Shifts (BIRDS) which originate from these agents (but exclude water) can provide temperature maps with good accuracy. While TmDOTP^5? emanates three major distinct proton resonances which are differentially sensitive to temperature and pH, TmDOTMA^? has a dominant pH‐insensitive proton resonance from a ? CH₃ group to allow higher signal‐to‐noise ratio (SNR) temperature assessment. Temperature (and pH) sensitivities of these resonances are practically identical at low (4.0T) and high (11.7T) magnetic fields and at nominal repetition times only marginal SNR loss is expected at the lower field. Since these resonances have extremely short relaxation times, high‐speed chemical shift imaging (CSI) is needed to detect them. Repeated in vivo CSI scans with BIRDS demonstrate excellent measurement stability. Overall, results with TmDOTP^5? and TmDOTMA^? suggest that BIRDS can be reliably applied, either at low or high magnetic fields, for functional studies in rodents. Copyright © 2009 John Wiley & Sons, Ltd.     

Assessment of human brain temperature by 1H MRS during visual stimulation and hypercapnia

Brain temperature is determined by the interplay between the cerebral metabolic rate of oxygen (CMRO2) and cerebral blood flow (CBF). In this study, single-voxel 1H nuclear MRS, with an accuracy of +/-0.2 degrees C for temperature determination, was used at 3 T to measure human brain temperature during visual stimulation (which increases both CBF and CMRO2) and hypercapnia (which increases CBF only). Visual stimulation had no detectable effect on brain temperature in the parenchyma showing blood oxygenation level dependent activation. Hypercapnia, leading to an increase in the end tidal CO2 by 8 +/- 2 mm Hg above the baseline, caused a short-lasting decrease in brain temperature of 0.30 +/- 0.33 degrees C. These results indicate that increased CBF may be a key factor, bringing about a small decrease in brain temperature during brain activation. However, the increase in CBF is not sufficient to lower brain temperature in the presence of a concomitant increase in endogenous heat production.     

Correlation between the temperature dependence of intrinsic MR parameters and thermal dose measured by a rapid chemical shift imaging technique

In order to investigate simultaneous MR temperature imaging and direct validation of tissue damage during thermal therapy, temperature‐dependent signal changes in proton resonance frequency (PRF) shifts, R₂* values, and T1‐weighted amplitudes are measured from one technique in ex vivo tissue. Using a multigradient echo acquisition and the Stieglitz‐McBride algorithm, the temperature sensitivity coefficients of these parameters are measured in each tissue at high spatiotemporal resolutions (1.6x1.6x4mm3, ≤ 5sec) at the range of 25‐61 °C. Non‐linear changes in MR parameters are examined and correlated with an Arrhenius rate dose model of thermal damage. Using logistic regression, the probability of changes in these parameters is calculated as a function of thermal dose to determine if changes correspond to thermal damage. Temperature sensitivity of R₂* and, in some cases, T1‐weighted amplitudes are statistically different before and after thermal damage occurred. Significant changes in the slopes of R₂* as a function of temperature are observed. Logistic regression analysis shows that these changes could be accurately predicted using the Arrhenius rate dose model (Ω = 1.01 ± 0.03), thereby showing that the changes in R₂* could be direct markers of protein denaturation. Overall, by using a chemical shift imaging technique with simultaneous temperature estimation, R₂* mapping and T1‐W imaging, it is shown that changes in the sensitivity of R₂* and, to a lesser degree, T1‐W amplitudes are measured in ex vivo tissue when thermal damage is expected to occur. These changes could possibly be used for direct validation of thermal damage in contrast to model‐based predictions. Copyright © 2011 John Wiley & Sons, Ltd.     

Skin blood flow in humans as a function of environmental temperature measured by ultrasound

We have measured the blood velocities in arteries supplying the skin in humans both in areas with arteriovenous anastomoses (AVA) and in skin areas without AVA in different temperature situations by means of a pulsed, bidirectional doppler ultrasound instrument. The instrument measures the time average of the instantaneous mean of the blood velocities present in a cross-section of a vessel. So long as the cross-sectional area of the vessel is constant, this average velocity is proportional to volume flow. We observe rapid and large fluctuations in blood velocities in arteries supplying skin areas with AVA in a comfortably warm environment. These fluctuations are substantially larger and more rapid than described by previous authors who have used plethysmograph methods. The blood velocities are more constant both at higher and lower environmental temperatures. There are no similar fluctuations in blood velocity in arteries supplying skin areas without AVA.     

A lanthanide complex with dual biosensing properties: CEST (chemical exchange saturation transfer) and BIRDS (biosensor imaging of redundant deviation in shifts) with europium DOTA-tetraglycinate

Responsive contrast agents (RCAs) composed of lanthanide(III) ion (Ln3R) complexes with a variety of1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (DOTA4S) derivatives have shown great potential as molecular imaging agents for MR. A variety of LnDOTA–tetraamide complexes have been demonstrated as RCAs for molecular imaging using chemical exchange saturation transfer (CEST). The CEST method detects proton exchange between bulk water and any exchangeable sites on the ligand itself or an inner sphere of bound water that is shifted by a paramagnetic Ln3R ion bound in the core of the macrocycle. It has also been shown that molecular imaging is possible when the RCA itself is observed (i.e. not its effect on bulk water) using a method called biosensor imaging of redundant deviation in shifts (BIRDS). The BIRDS method utilizes redundant information stored in the nonexchangeable proton resonances emanating from the paramagnetic RCA for ambient factors such as temperature and/or pH.Thus, CEST and BIRDS rely on exchangeable and nonexchangeable protons, respectively, for biosensing. We posited that it would be feasible to combine these two biosensing features into the same RCA (i.e. dual CEST and BIRDS properties). A complex between europium(III) ion (Eu3R) and DOTA–tetraglycinate [DOTA–(gly)S4] was used to demonstrate that its CEST characteristics are preserved, while its BIRDS properties are also detectable. The in vitro temperature sensitivity of EuDOTA–(gly)S4 was used to show that qualitative MR contrast with CEST can be calibrated using quantitative MR mapping with BIRDS, thereby enabling quantitative molecular imaging at high spatial resolution.     

Effects of temperature on intracellular sodium, pH and cellular energy status in RIF-1 tumor cells

Most perfused tumor cell experiments are performed at 37 degrees C, the normal healthy body temperature. However, the temperature of subcutaneously implanted tumors in small animals is generally 29-33 degrees C when the rectal temperature of the animal is maintained at 37 degrees C. We have investigated the acute effects of increasing the temperature of perfused radiation-induced-fibrosarcoma (RIF-1) tumor cells from 33 to 37 degrees C (30 min) on intracellular sodium (Na(i)+) , intracellular pH (pH(i)), and bioenergetic status. Heating the cells by 4 degrees C produced a reversible increase in Na(i)+, slight acidification and no change in nucleotide triphosphate to inorganic phosphate ratio (NTP/P(i)) as measured by shift-reagent-aided (23)Na and (31)P NMR spectroscopy. In the presence of 3 microM 5-(N-ethyl-N-isopropyl) amiloride (EIPA), a potent and specific inhibitor of Na(+)/H(+) antiporter, the increase in Na(i)+ during the heating was completely abolished suggesting that the heat induced increase in Na(i)+ was caused by an increase in Na(+)/H(+) antiporter activity. However, the changes in pH(i) with the heating were identical with or without EIPA, indicating that pH(i) is controlled by other ion exchange mechanisms in addition to Na(+)/H(+) antiporter. NTP/P(i) was significantly higher in presence of EIPA for some time points during the heating suggesting that both NTP production and consumption rates may be altered during the heating. These results indicate that a slight increase in temperature from 33 to 37 degrees C induces significant changes in Na(+) physiology largely because of activation of Na(+)/H(+) antiporter but other ion exchange mechanisms are also involved in maintaining pH(i) in the RIF-1 tumor cells. Thus, care must be taken in choosing the temperature for perfused cell studies.     

Regulation of intracellular sodium and pH by the electrogenic H+ pump in frog skin

We have investigated the role of the electrogenic hydrogen ion pump in the regulation of intracellular sodium ion activity (a _Na ⁱ ) and intracellular pH (pH_i) in frog skin epithelial cells using double-barreled ion sensitive microelectrodes. WhenRana esculenta skin is mounted in an Ussing chamber and bathed in 1 mM Na₂SO₄ buffered to pH 7.34 with imidazole on the apical side and in normal Ringer on the serosal side, the apical addition of the carbonic anhydrase inhibitor, ethoxzolamide (10^–4M) blocks net H⁺ ion excretion and Na absorption, producing a depolarization of 25–30 mV of the apical membrane, potential (mc). We demonstrate the these changes are accompanied by a fall ina _Na ⁱ from 6.2±0.5 mmol/l to 3.4±0.6 mmol/l and an increase in pH_i from 7.20±0.03 to 7.38±0.08 (n=12 skins). Voltage clamping mc to its control value in the presence of ethoxzolamide restoreda _Na ⁱ but the pH_i remained alkaline. Furthermore, the fall ina _Na ⁱ produced by ethoxzolamide could be mimicked by voltage clamping mc towards the value of the Nernst potential for Na at the apical membrane. These results indicate that the maintenance of the cellular Na⁺ transport pool is dependent on a favourable electrical driving force and counter-current generated by an electrogenic H⁺ pump at the apical membrane.Addition of amiloride (10^–5 mol/l) or substitution of external Na⁺ by Mg²⁺ or K⁺ caused a hyperpolarization of mc and a fall ina _Na ⁱ . These effects were accompanied by an inhibition of H⁺ excretion and a fall in pH_i of 0.14 ±0.08 units (n=6 skins). However, when the effect, of Na⁺ transport inhibition on mc was prevented by imposing a voltage clamp no effects of amiloride on H⁺ excretion or pH_i were observed. Moreover, the effect of amiloride on pH_i could be reproduced in control skins by voltage clamping mc to –100 mV. The metabolic inhibitors vanadate (1 mmol/l) and di-cyclo hexyl carbodiimide (5×10^–5 mol/l) inhibited H⁺ excretion and decreased pH_i from 7.28±0.08 to 7.02±0.06 and from 7.30±0.06 to 7.12±0.05 (n=6 skins), respectively.These results indicate that an apical membrane H⁺ ATPase plays a role in regulating pH_i and the mechanism is sensitive to membrane potential.     

Immunoproteasome in the blood plasma of children with acute appendicitis,and its correlation with proteasome and UCHL1 measured by SPR imaging biosensors

The aim of this study was to determinate the immunoproteasome concentration in the blood plasma of children with appendicitis, and its correlation with circulating proteasome and ubiquitin carboxyl‐terminal hydrolase L1 (UCHL1). Twenty‐seven children with acute appendicitis, managed at the Paediatric Surgery Department, were included randomly into the study (age 2 years 9 months up to 14 years, mean age 9·5 ± 1 years). There were 10 girls and 17 boys; 18 healthy, age‐matched subjects, admitted for planned surgeries served as controls. Mean concentrations of immunoproteasome, 20S proteasome and UCHL1 in the blood plasma of children with appendicitis before surgery 24 h and 72 h after the appendectomy were higher than in the control group. The immunoproteasome, 20S proteasome and UCHL1 concentrations in the blood plasma of patients with acute appendicitis were highest before surgery. The immunoproteasome, 20S proteasome and UCHL1 concentration measured 24 and 72 h after the operation decreased slowly over time and still did not reach the normal range (P < 0·05). There was no statistical difference between immunoproteasome, 20S proteasome and UCHL1 concentrations in children operated on laparoscopically and children after classic appendectomy. The immunoproteasome concentration may reflect the metabolic response to acute state inflammation, and the process of gradual ebbing of the inflammation may thus be helpful in the assessment of the efficacy of treatment. The method of operation – classic open appendectomy or laparoscopic appendectomy – does not influence the general trend in immunoproteasome concentration in children with appendicitis.     

Doubly selective multiple quantum chemical shift imaging and T1 relaxation time measurement of glutathione (GSH) in the human brain in vivo

Mapping of a major antioxidant, glutathione (GSH), was achieved in the human brain in vivo using a doubly‐selective multiple quantum filtering based chemical shift imaging (CSI) of GSH at 3 T. Both in vivo and phantom tests in CSI and single voxel measurements were consistent with excellent suppression of overlapping signals from creatine, γ‐Amino butyric acid (GABA) and macromolecules. GSH concentration in the fronto‐parietal region was 1.20 ± 0.16 µmol/g (mean ± SD, n = 7). The longitudinal relaxation time (T₁) of GSH in the human brain was 397 ± 44 ms (mean ± SD, n = 5), which was substantially shorter than that of other metabolites. This GSH‐CSI method permits us to address regional differences of GSH in the human brain under conditions where oxidative stress has been implicated, including multiple sclerosis, aging and neurodegenerative diseases. Copyright © 2012 John Wiley & Sons, Ltd.     

Interstitial PCO2 and pH in rat hippocampal slices measured by means of a novel fast CO2/H+-sensitive microelectrode based on a PVC-gelled membrane

We describe here the construction and properties of a double-barrelled microelectrode (tip diameter 4–10 m) which permits simultaneous measurements of PCO₂ and pH, and which has a 90% response time of only one or a few seconds for a step change in PCO₂. The fast response of the CO₂-sensitive barrel is due to (i) the use of a PVC-gelled (tridodecylamine-containing) membrane solution which enables the construction of extremely short ( 4 m), yet mechanically stable, membrane columns, and (ii) the presence of carbonic anhydrase in the filling solution. Recordings made in the pyramidal layer of area CA1 in rat hippocampal slices showed that the deviation in the acid direction of the basal interstitial pH (pH₀) from that of the perfusion solution was attributable to a higher PCO₂ level within the tissue. Most of the late acid shift evoked by stimulation of the Schaffer collaterals (5- to 20-s trains at 10 Hz) could also be explained on the basis of an accumulation of interstitial CO₂ at a constant HCO ₃ ^– concentration. This conclusion was supported by the finding that inhibition of extracellular carbonic anhydrase activity by 10 M benzolamide completely abolished the activity-induced fall in pH₀, but not the increase in PCO₂. The initial stimulus-induced alkalosis was accompanied by a slight decrease in PCO₂ only, implying a parallel increase in the interstitial HCO ₃ ^– concentration. Benzolamide produced a dramatic enhancement of the early alkaline shift as well as of the simultaneous fall in PCO₂. The latter effect of the drug unmasks a cellular CO₂ sink that is induced by neuronal activity.     

Imaging the extracellular pH of tumors by MRI after injection of a single cocktail of T1 and T2 contrast agents

The extracellular pH (pH(e) ) of solid tumors is acidic, and there is evidence that an acidic pH(e) is related to invasiveness. Herein, we describe an MRI single-infusion method to measure pH(e) in gliomas using a cocktail of contrast agents (CAs). The cocktail contained gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaminophosphonate (GdDOTA-4AmP) and dysprosium-1,4,7,10-tetraazacyclododecane-N,N',N',N'-tetrakis(methylenephosphonic acid) (DyDOTP), whose effects on relaxation are sensitive and insensitive to pH, respectively. The Gd-CA dominated the spin-lattice relaxivity ΔR(1) , whereas the Dy-CA dominated the spin-spin relaxivity ΔR(2)*. The ΔR(2)* effects were used to determine the pixel-wise concentration of [Dy] which, in turn, was used to calculate a value for [Gd] concentration. This value was used to convert ΔR(1) values to the molar relaxivity Δr(1) and, hence, pH(e) maps. The development of the method involved in vivo calibration and measurements in a rat brain glioma model. The calibration phase consisted of determining a quantitative relationship between ΔR(1) and ΔR(2)* induced by the two pH-independent CAs, gadolinium-diethylenetriaminepentaacetic acid (GdDTPA) and DyDOTP, using echo planar spectroscopic imaging (EPSI) and T(1) -weighted images. The intensities and linewidths of the water peaks in EPSI images were affected by CA and were used to follow the pharmacokinetics. These data showed a linear relationship between inner- and outer-sphere relaxation rate constants that were used for CA concentration determination. Nonlinearity in the slope of the relationship was observed and ascribed to variations in vascular permeability. In the pH(e) measurement phase, GdDOTA-4AmP was infused instead of GdDTPA, and relaxivities were obtained through the combination of interleaved T(1) -weighted images (R(1) ) and EPSI for ΔR(2)*. The resulting r(1) values yielded pH(e) maps with high spatial resolution.     

Vascular effects of endothelin-1 in the cat; modification by indomethacin and l-NAME

Intravenous infusion of endothelin-1 (ET-1) in the cat, 60 pmol × kg body wt^-1x min^-1for 5 min, induced an increase in mean arterial blood pressure (MAP) of 41.3 ± 4.8 mmHg (n= 6; P < 0.001). Blood flow, as determined with radioactive microspheres, was reduced in many tissues. Reductions by 70–80% were observed in the choroid plexus, pineal and pituitary glands. Total cerebral blood flow was reduced by 18–23%. Pre-treatment with indomethacin or a combination of indomethacin and l -NAME caused vasoconstriction in many tissues and modified the responses to ET-1 in a variable way, suggesting that normally, ET-1 tends to release arachidonic acid metabolites and nitric oxide with great variations between different tissues. Intracerebroventricular infusion (i.c.v.) of ET-1, 10 pmol × kg body wt^-1x min^-1, caused an increase in MAP of 79 ± 11 mmHg (n= 6; P < 0.001). Regional blood flow in the medulla oblongata, medulla spinalis, choroid plexus, pineal and pituitary glands was reduced by 60–80%. Heart rate, cardiac output and coronary blood flow were significantly increased after 30 min i.c.v. infusion, indicating an activation of the heart, most probably as part of a central ischaemic response. Our results indicate that in many tissues the vasoconstrictive effect of ET-1 is influenced by indomethacin- and l -NAME-sensitive vasodilator mechanisms that are activated by the peptide. In the CNS, there may be marked effects on regional blood flow after i.c.v. infusion.     

Multi‐echo susceptibility‐weighted imaging and histology of open‐field blast‐induced traumatic brain injury in a rat model

Blast‐induced traumatic brain injury is on the rise, predominantly as a result of the use of improvised explosive devices, resulting in undesirable neuropsychological dysfunctions, as demonstrated in both animals and humans. This study investigated the effect of open‐field blast injury on the rat brain using multi‐echo, susceptibility‐weighted imaging (SWI). Multi‐echo SWI provided phase maps with better signal‐to‐noise ratio (SNR) and contrast‐to‐noise ratio (CNR), making it a sensitive technique for brain injury. Male Sprague‐Dawley rats were subjected to a survivable blast of 180 kPa. The visibility of blood vessels of varying sizes improved with multi‐echo SWI. Reduced signal intensity from major vessels post‐blast indicates increased deoxyhaemoglobin. Relative cerebral blood flow was computed from filtered phase SWI images using inferred changes in oxygen saturation from major blood vessels. Cerebral blood flow decreased significantly at day 3 and day 5 post‐blast compared with that pre‐blast. This was substantiated by the upregulation of β‐amyloid precursor protein (β‐APP), a marker of ischaemia, in the neuronal perikaya of the cerebral cortex, as observed by immunofluorescence, and in the cortical tissue by western blot analysis. Our findings indicate the presence of brain ischaemia in post‐blast acute phase of injury with possible recovery subsequently. Our results from cerebrovascular imaging, histology and staining provide an insight into the ischaemic state of the brain post‐blast and may be useful for prognosis and outcome. Copyright © 2015 John Wiley & Sons, Ltd.     

Early detection of radiation therapy response in non‐Hodgkin's lymphoma xenografts by in vivo 1H magnetic resonance spectroscopy and imaging

The purpose of the study was to investigate the capability of ¹H MRS and MRI methods for detecting early response to radiation therapy in non‐Hodgkin's lymphoma (NHL). Studies were performed on the WSU‐DLCL2 xenograft model in nude mice of human diffuse large B‐cell lymphoma, the most common form of NHL. Radiation treatment was applied as a single 15 Gy dose to the tumor. Tumor lactate, lipids, total choline, T₂ and apparent diffusion coefficients (ADC) were measured before treatment and at 24 h and 72 h after radiation. A Hadamard‐encoded slice‐selective multiple quantum coherence spectroscopy sequence was used for detecting lactate (Lac) while a stimulated echo acquisition mode sequence was used for detection of total choline (tCho) and lipids. T₂‐ and diffusion‐weighted imaging sequences were used for measuring T₂ and ADC. Within 24 h after radiation, significant changes were observed in the normalized integrated resonance intensities of Lac and the methylenes of lipids. Lac/H₂O decreased by 38 ± 15% (p = 0.03), and lipid (1.3 ppm, CH₂)/H₂O increased by 57 ± 14% (p = 0.01). At 72 h after radiation, tCho/H₂O decreased by 45 ± 14% (p = 0.01), and lipid (2.8 ppm, polyunsaturated fatty acid)/H₂O increased by 970 ± 36% (p = 0.001). ADC increased by 14 ± 2% (p = 0.003), and T₂ did not change significantly. Tumor growth delay and regression were observed thereafter. This study enabled comparison of the relative sensitivities of various ¹H MRS and MRI indices to radiation and suggests that ¹H MRS/MRI measurements detect early responses to radiation that precede tumor volume changes. Copyright © 2010 John Wiley & Sons, Ltd.     

Blood flow in histamine- and allergen-induced weal and flare responses, effects of an H1 antagonist, α-adrenoceptor agonist and a topical glucocorticoid

Allergen has previously been shown to induce a continuous increase in local dermal blood flow after a prick test in allergic subjects, whereas histamine induced, initially, similar peak increases in blood flow of much shorter duration. Blood flow changes induced by histamine and allergen have now been evaluated (i) after pretreatment with a local corticosteroid cream, clobetasole-17-propionate; (ii) after oral administration of the H1-antihistamine loratadine; and (iii) after oral pretreatment with the alpha 1-adrenoceptor agonist pseudoephedrine. Blinded placebo-controlled designs were used in the substudies. Laser doppler flowmetry was used for non-invasive recording of changes in local blood flow intermittently for 24 h after the topical corticosteroid, 6 h for the substudies on loratadine and pseudoephedrine. The size of the immediate weal and flare reactions, as well as late phase reactions, were also determined. Pretreatment with clobetasole-17-propionate cream on the skin for 1 week prior to prick tests did not affect the blood flow response elicited by histamine or allergen, in either the initial part (up to 1 h) or the protracted 24 h determinations. The size of the weal and flare reactions decreased. Loratadine and pseudoephedrine did not reduce the initial allergen-induced increase in blood flow, while lower blood flow compared with placebo pretreatment was noted for the protracted (1-6 h) determinations. Blood flow changes after histamine were unaffected. The histamine-induced weal and flare was inhibited by loratadine more effectively than the corresponding allergen-induced reaction. The weal and flare reactions after histamine and allergen were not changed after pseudoephedrine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Micellization Kinetics of a Novel Multi‐Responsive Double Hydrophilic Diblock Copolymer Studied by Stopped‐Flow pH and Temperature Jump

Double hydrophilic diblock copolymer, poly(N‐isopropylacrylamide)‐block‐poly(2‐diethylamino ethyl methacrylate) (PNIPAM‐b‐PDEA), was synthesized via reversible addition‐fragmentation chain transfer (RAFT) polymerization. Containing the well‐known thermo‐responsive PNIPAM block and pH‐responsive PDEA block, this novel diblock copolymer exhibits intriguing “schizophrenic” micellization behavior in aqueous solution, forming PDEA‐core micelles at alkaline pH and room temperature, and PNIPAM‐core micelles at acidic pH and elevated temperatures. The kinetics of the pH‐ and thermo‐responsive micellization processes were studied in detail using a stopped‐flow apparatus equipped with a newly developed millisecond temperature jump (mT‐jump) accessory. Upon a pH jump from 4 to 12 at 25 °C, the early stages of relaxation curves monitoring the formation PDEA‐core micelles can be well‐fitted using a double‐exponential function, leading to two characteristic relaxation time constants, τ₁ and τ₂. As τ₂ decreases with increasing polymer concentration, the slow process is thus expected to proceed via micelle fusion/fission mechanism, approaching the final equilibrium state. Upon a temperature jump from 20 to 45 °C at pH 4, the relaxation curves monitoring the formation PNIPAM‐core micelles can also be well‐fitted using a double‐exponential function. The fast process (τ₁) is associated with the quick association of unimers into a large amount of small micelles and the formation of quasi‐equilibrium micelles. τ₂ is almost independent of polymer concentration, suggesting that unimer insertion/expulsion is the main mechanism for the slow process. The protonated PDEA corona of quasi‐equilibrium micelles renders the micelle fusion/fission mechanism less favorable due to electrostatic repulsion.

     

Detection and quantification of VEGFR-1 in the nuclei of tumor cells by a new flow cytometry-based method

Flow cytometry using fluorescent antibodies (FC) is the method of choice for the quantitation of proteins expressed at the surface or inside the cell, but, however, does not allow to selectively measure nuclear expression. We therefore sought to develop a method for the extraction of intact cell nuclei, which can be used for their subsequent immunofluorescent analysis by FC. The studied protein was vascular endothelial growth factor-receptor-type 1 (VEGFR-1) which is important in tumor survival and metastasis. Two human cell lines, A431 (epidermoid carcinoma of skin with low invasive and metastatic potential) and BRO (highly aggressive amelanotic melanoma), were used as examples for tumor cells, and normal human fibroblasts PHF served as a control line. The quality of the extracted nuclei was assessed by their intactness and purity from cytoplasm. The high content of the nuclear markers (PCNA?=?proliferating cell nuclear antigen, lamin A/C) in the extracted nuclei with almost complete absence of the cytoplasmic β-tubulin demonstrated that the protocol can be used to obtain a pure suspension of single intact cell nuclei. The measurement of the nuclear VEGFR-1 content revealed that it was present only in tumor cell nuclei and that in more malignant BRO cells the receptor content was 1.75 times higher than in A431 (p?=?0.014). Thus, the developed method of extraction of cell nuclei for subsequent FC analysis is suitable for the quantitative evaluation of protein content in the native nucleus.