Development of a tissue-engineered composite implant for treating traumatic paraplegia in rats.

This study was designed to assess a new composite implant to induce regeneration of injured spinal cord in paraplegic rats following complete cord transection. Neuronal xenogeneic cells from biopsies of adult nasal olfactory mucosa (NOM) of human origin, or spinal cords of human embryos, were cultured in two consecutive stages: stationary cultures in a viscous semi-solid gel (NVR-N-Gel) and in suspension on positively charged microcarriers (MCs). A tissue-engineered tubular scaffold, containing bundles of parallel nanofibers, was developed. Both the tube and the nanofibers were made of a biodegradable dextran sulphate-gelatin co-precipitate. The suturable scaffold anchored the implant at the site of injury and provided guidance for the regenerating axons. Implants of adult human NOM cells were implanted into eight rats, from which a 4 mm segment of the spinal cord had been completely removed. Another four rats whose spinal cords had also been transected were implanted with a composite implant of cultured human embryonic spinal cord cells. Eight other cord-transected rats served as a control group. Physiological and behavioral analysis, performed 3 months after implantation, revealed partial recovery of function in one or two limbs in three out of eight animals of the NOM implanted group and in all the four rats that were implanted with cultured human embryonic spinal cord cells. Animals of the control group remained completely paralyzed and did not show transmission of stimuli to the brain. The utilization of an innovative composite implant to bridge a gap resulting from the transection and removal of a 4 mm spinal cord segment shows promise, suggesting the feasibility of this approach for partial reconstruction of spinal cord lesions. Such an implant may serve as a vital bridging station in acute and chronic cases of paraplegia.

TNFalpha stimulated ATP-sensitive potassium channels and attenuated deoxyglucose and Ca uptake of H9c2 cardiomyocytes.

Tumor necrosis factor (TNFalpha) is an inflammatory cytokine that induces programmed cell death in a variety of tissue types, including the heart. Recent experimental data suggest that the TNF expressed within the myocardium in response to environmental injury plays an important role in initiating homeostatic response. The effect of TNFalpha (10-50 ng/mL) was studied on (86)Rb efflux, (3)H-deoxyglucose uptake, or (45)Ca uptake in H9c2 cardiomyocytes. TNFalpha stimulated (86)Rb efflux from cultures, while 2 micro M glibenclamide blocker of ATP-sensitive potassium channels or 20 micro M zvad-fmk caspase inhibitor attenuated this effect. TNFalpha also depressed the stimulatory effect of 80 mM KCl on (45)Ca uptake in the cardiomyocytes. TNFalpha inhibited the stimulatory effect of 100 nM insulin on (3)H-deoxyglucose uptake. Our findings further suggest that TNFalpha mediated adaptive and protective effects in the heart during a brief environmental injury.

Simvastatin induces apoptosis of cultured rat cardiomyocytes.

Considering the therapeutic effect of statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) and simvastatin in patients with coronary heart disease, our first hypothesis was that simvastatin should inhibit apoptosis (programmed cell death) in angiotensin II-treated cultured myocytes. But after realizing that simvastatin stimulates apoptosis, we changed our hypothesis and began to study its apoptotic effect in primary cultured rat cardiomyocytes. We found that simvastatin induced apoptosis in a dose-dependent manner (0.1 to 3 micromol/L), as evidenced by the appearance of increased DNA fragmentation in agarose gels and characteristic apoptotic patterns in nuclei labeled with Hoechst 33342, as well as increased activity of caspase 3. FACS analysis of simvastatin-treated cardiomyocytes showing annexin V binding and propidium iodide exclusion ruled out the possibility of necrosis. Increased intracellular enzymatic activity of creatine phosphokinase, aldolase, and lactic dehydrogenase, markers for normal cell function, could reflect the hypertrophic effect of simvastatin. The results indicate that simvastatin-induced apoptosis in cultured heart cells is concentration-dependent and additive to the apoptotic effect of angiotensin II.

Uptake of glucose analogs reflects the rate of contraction of cultured myocytes.

The present study demonstrates that: a) adenosine and R-N6-(2-phenylisopropyl)-adenosine (R-PIA, A1 and A3 adenosine receptor agonist) inhibited [3H]deoxyglucose uptake or [3H]3-O-methyl-D-glucose uptake; b) sugar uptake reflects the rate of contraction in cardiac cultures; c) [3H]deoxyglucose uptake or [3H]3-O-methyl-D-glucose uptake are useful quantitative probes for beating rate evaluation. A 25-40% decrease in [3H]deoxyglucose uptake (p < 0.01) was obtained following 13-21 min treatment with 100 microM adenosine together with 1 microM dipyridamole or with 10 microM R-PIA, which inhibited spontaneous contractions. Adenosine (10 microM) attenuated spontaneous beating rate and inhibited approximately 55% of the [3H]deoxyglucose uptake following 22 h treatment (p < 0.01). 1 microM R-PIA also attenuated beating rate following either a short (1 min) or long (24 h) application and decreased [3H]deoxyglucose uptake by 20-30% (p < 0.01) during 0.5-24 h of treatment. A 157 +/- 9% and 205 +/- 11% increase (p < 0.01) in [3H]deoxyglucose uptake was obtained at 27 and 37 degrees C, respectively, compared with the uptake at 17 degrees C, which completely inhibited spontaneous contractions. Similar results [33 +/- 6% (p < 0.01) and 21 +/- 8% (p < 0.05) inhibition in [3H]deoxyglucose uptake] were obtained following 2 and 22 h of carbamylcholine treatment, respectively. This treatment also reduced spontaneous contractions. [3H] 3-O-Methyl-D-glucose uptake also decreased by 31 +/- 12% (p < 0.05) as a result of the arrest of contractions by adenosine. Elevations of 90 +/- 13% and 34 +/- 11% (p < 0.01) in [3H]deoxyglucose uptake were obtained following treatment with isoprenaline after 2 and 22 h application, respectively. It is concluded that adenosine and R-PIA inhibited [3H]deoxyglucose uptake or [3H] 3-O-methyl-D-glucose uptake in rat heart culture and that there is a linkage between the rate of cardiac contractions in culture and sugar uptake.

Regulation of A1 adenosine receptors by amiodarone and electrical stimulation in rat myocardial cells in vitro.

The effects of conditions that either increase or decrease heart rate on the pharmacological properties of adenosine receptors in cultured rat myocytes were examined. Levels of A1 adenosine receptors, following prolonged treatment with electrical stimulation (ES) or the antiarrhythmic drug amiodarone, were determined using radioligand binding with the specific A1 receptor antagonist [3H]1,3-dipropyl-8-cyclopentylxanthine (CPX). The effects of lowering temperature were also explored. Exposure to amiodarone for 4 days reduced the density of A1 receptors by 19% (from 24.7 +/- 0.4 to 20.09 +/- 0.3 fmol/dish) and inhibited the rate of contraction by 60% (from 188 +/- 16 to 76 +/- 30 beats/min), without changing the receptor affinity, protein content, creatine kinase (CK) activity or cell number. Electrical stimulation at 25 degrees C elevated the density of A1 adenosine receptors by 185% (from 4.1 +/- 0.4 to 11.69 +/- 2.1 fmol/dish). Four days of reduced temperature (from 37 degrees C to either 30 or 25 degrees C) lowered the density of A1 adenosine receptors by 69 or 86%, respectively (from 24.1 +/- 1.2 to 7.4 +/- 0.4 or 3.4 +/- 0.3 fmol/dish), with no significant change in the receptor affinity, activity of CK, or lactate dehydrogenase (LDH), protein content or cell number. The observed up- and down-regulation of A1 adenosine receptors in primary myocyte cultures in response to conditions that exogenously alter the rate of contraction, is indicative of the role of adenosine receptors in adaptation of heart cells to stress.

Effects of theophylline and dibutyryl-cAMP on adenosine receptors and heart rate in cultured cardiocytes.

The effects of chronic exposure to the adenosine antagonist theophylline (Theo) and dibutyryl cyclic-AMP, a membrane-permeant derivative of the second messenger 3', 5'-cyclic-AMP (cAMP), on contractions and adenosine receptor levels in cultured cardiocytes were studied. Binding of the A1-adenosine receptor antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]CPX) was used to monitor the level of the receptors in intact cardiocytes. Both Theo and cAMP stimulated the rate of contraction and also increased the density of adenosine receptors. The Bmax value for [3H]CPX binding to intact cardiocytes was increased by 45-47% following 4 days of exposure to either 50 microM Theo or 100 microM cAMP. Scatchard analysis indicated that the affinity of the A1 receptors for [3H]CPX remained unchanged (Kd 0.1-0.2 nM). No significant differences were observed in protein content or in cell number. A linear correlation was achieved between the level of A1-adenosine receptors and heart rate at various Theo and dibutyryl-cAMP concentrations, although Theo was more efficient in elevation of the receptor density. Increases of 82, 78, 138 and 235% in A1 receptor density and increases of 63, 59, 66 and 150% in heart rate were obtained following 5 days of treatment with 1, 10, and 1000 microM of Theo, respectively. It is concluded that there is a linkage between the rate of cardiac contractions and the level of adenosine receptors. Thus, changes in the density of adenosine receptors may compensate for chronic drug-induced changes in cardiac contractile activity so as to restore conditions to the normal state.

Regulation of adenosine receptors in cultured heart cells.

A1 adenosine receptors were studied on heart cells grown in cultures by the radioligand binding technique. Treatments with agents that accelerated heart rate for 3-4 days, caused an increase in the level of adenosine receptors. Treatments that attenuated heart rate, reduced the level of the receptors. Thus, the cardiac cells respond to environmental conditions affecting heart contraction so as to restore the basal rate of heart activity.

Characterization of adenosine receptors in intact cultured heart cells.

Adenosine receptors were studied on heart cells grown in cultures by the radioligand binding technique. We used the hydrophilic A1 adenosine receptor radioligand [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]CPX), to monitor the level of the receptors on intact cardiocytes. The binding showed high affinity (Kd = 0.13 nM) and the number of [3H]CPX binding sites (Bmax) was 23.1 fmol/dish (21 fmol/mg protein). The Ki of the agonists R-N6-(2-phenylisopropyl)-adenosine (R-PIA) and S-N6-(2-phenylisopropyl)-adenosine (S-PIA), and of the antagonists CPX and theophylline were 3.57, 49.0, 1.63 and 4880 nM, respectively. The number of adenosine receptors was very low during the first days in cultures (5 fmol/dish) and increased gradually until it reached a plateau on days 8-10. Treatment with norepinephrine or isoproterenol which accelerated the rate of contractions, induced up regulation of the receptors. Bmax increased 2-3-fold by application of norepinephrine for 4 days, while receptor affinity to the radioligand was unaffected. Lactate dehydrogenase (LDH) and creatine kinase (CK) activity increased only by 22 and 38%, respectively. Similarly, 3 days treatment with triiodothyronine (T3, 10(-8) M), which also accelerated heart rate, increased the number of adenosine receptors by 56% without a significant change in the affinity of the receptors to [3H]CPX. Carbamylcholine (5 x 10(-6) M), which reduced the rate of heart contractions, caused 26% down regulation while the affinity of the receptors remained unchanged. It is concluded that there is a linkage between the rate of cardiac contractions and the level of adenosine receptors. Thus, the level of adenosine receptors may respond to drug-induced chronic changes in cardiac contractile activity so as to restore conditions to normal (basal) contractions.