We have examined the cellular distribution of both FGF-2 and FGFR1 immunoreactivity and their mRN... more We have examined the cellular distribution of both FGF-2 and FGFR1 immunoreactivity and their mRNAs throughout the normal adult rat brain in order to reconcile numerous disparate findings in the published literature. The results confirm a widespread distribution of FGF-2 and FGFR1 in the rat brain, and different regions express distinct patterns of FGF-2 and FGFR1 mRNA and protein: neuronal and non-neuronal cells show different subcellular distributions that vary according to the area where they are located. The intensity of the staining and hybridization also varies according to the loci examined and the cell type involved. Astrocytes contain the highest levels of FGF-2 and FGFR1 mRNAs, and characteristically, possess high levels of immunoreactive FGF-2 within the nucleus. Amongst non-neuronal cells, oligodendrocytes do not synthesize or contain significant levels of FGF-2 immunoreactivity however, they do express FGFR1 mRNA. In these cells, immunoreactive FGFR1 is mainly associated with the myelin sheaths of neuronal fibers. In ventricular systems, ependymal cells synthesize and contain immunoreactive FGFR1. In contrast, only cells lining the lateral wall of the IIIrd ventricle express FGF-2 mRNA. Subependymal cells contain high levels of both FGF-2 and FGFR1 immunoreactivity.Neurons express low levels of FGF-2 mRNA and immunoreactive FGF-2 is localized predominantly to the perikaryon. However, selected populations of neurons, such as CA2 field of the hippocampus, show high levels of FGF-2 mRNA, in which the nucleus is strongly immunopositive. Similarly, high levels of FGFR1 mRNA are localized to select populations of neurons (e.g. amygdala). FGFR1 immunoreactivity is mainly associated with myelinated fiber tracts (e.g. striatum), and some neurons show immunoreactivity in the perikaryon (e.g. hippocampus), the nucleus (e.g. mesencephalic trigeminal nucleus), or in axonal projections (e.g. hypothalamus). Remarkably, in many of the areas studied, FGF-2 and FGFR1 mRNA and/or their translated protein do not co-localize in neurons (e.g. neo-cortices) or even in the same regions of the brain (e.g. substantia nigra). In other instances, mRNAs for both FGF-2 and FGFR1 colocalize (e.g. supraoptic nucleus).The brain, in contrast to peripheral tissues, contains high levels of FGF-2 and actively expresses its gene under normal physiological conditions. The highly specific anatomical distribution of immunoreactive FGF-2 in neuronal and non-neuronal brain cells, supports the notion that it plays a multifunctional role in the CNS under normal physiology. By correlating the localization and the synthesis of FGF-2 and one of its high affinity receptors, FGFR1, in the CNS, it should be possible to obtain a better understanding of the roles of FGF-2 in normal and pathological conditions.
The single copy gene for human basic fibroblast growth factor (bFGF) has been shown to encode not... more The single copy gene for human basic fibroblast growth factor (bFGF) has been shown to encode not one but multiple proteins of 24, 23, 22 and 18 kD. Although bioactivities of the 18 kD protein are currently used to define bFGF gene function, it is not yet known if the three larger proteins have these same bioactivities or whether they will serve to define new bFGF gene functions. In this report we present a comparative study describing the de novo synthesis, transport, processing and intracellular location of individual bFGF isoforms. Data from cDNA mutagenesis and COS cell expression experiments show that individual isoforms are differentially localized to either the cell surface or to the nucleus. The 24, 23 and 22 kD proteins (CUG-mediated initiation) exclusively localize in the nucleus while the 18 kD protein (AUG-mediated initiation) is preferentially exported onto the cell surface, but is not released into the surrounding culture medium. Specific CUG or AUG translation initiation codons are necessary and sufficient for the synthesis of each isoform examined and thereby, indirectly, mediate differential localization. Since bFGF does not contain the characteristic signals predicted for cell surface or nuclear targeting, our continuing studies will either unmask its functionally equivalent domain(s) or will identify the requisite participation of yet unknown cellular components.
In the central nervous system (CNS), nerve regeneration after traumatic injury fails. The formati... more In the central nervous system (CNS), nerve regeneration after traumatic injury fails. The formation of a dense fibrous scar is thought to restrict in part the growth of axonal projections, providing one of the many reasons that complete lesions of neural pathways in the adult mammalian CNS are rarely followed by significant functional recovery. In order to determine which mechanisms mediate scar formation in the CNS and to investigate whether they can be modulated in vivo, we have attempted to define the potential role of trophic factors. Our previous studies have shown the focal elevation of transforming growth factor β1 (TGFβ1) expression in lesioned CNS tissue. In the studies described here, we demonstrate that TGFβ1 participates in the scarring response in the rat brain. First, the elevated protein levels of TGFβ1 are localized to specific populations of injury-responsive cells in the traumatized CNS. Furthermore, the injection of TGFβ1 into the brains of injured rats causes a dramatic increase in the scarring response. Conversely, when neutralizing TGFβ1 antibodies are administered, the deposition of fibrous scar tissue and the formation of a limiting glial membrane that borders the lesion is significantly attenuated, thus establishing a role for the endogenous growth factor in regulation of the non-glial component of the scar. In implicating TGFβ1 in the scarring response in the CNS, the potential use for TGFβ1 antagonists as inhibitors of scar formation in the injured mammalian CNS is self-evident.
The expression of basic FGF mRNA, while virtually absent in peripheral tissues, appears to be con... more The expression of basic FGF mRNA, while virtually absent in peripheral tissues, appears to be constitutively expressed in the central nervous system. As such, while it is difficult to detect any mRNA encoding basic FGF in the heart, lung, kidneys, ovaries, liver, or pituitary of rats, the levels are easily detected in brain. A regional analysis of the expression of basic FGF mRNA in brain reveals that it is widely distributed in the cortex (frontal, parietal, and occipital), the hippocampus, hypothalamus, and pons. Only a few loci of basic FGF synthesis are detected by in situ hybridization and include layers 2 and 6 of the medial (cingulate) cortex, the indusium griseum, fasciola cinereum, and field CA2 of the hippocampus. The identification of specific cell populations in the brain, and particularly in the hippocampus, that synthesize basic FGF supports the notion that this potent neurotrophic factor is involved in normal CNS function and that the presence (or absence) of its expression may be linked to the pathogenesis of the neurogenerative diseases characterizing these various loci. The significance of these findings with respect to the regulation of basic FGF expression in peripheral tissue and the central nervous system is discussed.
Bipolar radiofrequency (RF) ablation, especially with perfusion of saline, has been shown to incr... more Bipolar radiofrequency (RF) ablation, especially with perfusion of saline, has been shown to increase volume over monopolar conventional methods. The aims of this study are to study whether this method is linked to too flattened thermal lesions and premature rise of impedance and to elucidate some safety concerns. Eighteen RF ablations were performed using a 1.8-mm-diameter bipolar applicator in the liver of nine healthy pigs through laparotomy with or without temporary vascular occlusion [the Pringle maneuver (PGM)]: group A (n=9), without PGM; group B (n=9), with PGM. Hypertonic saline solutions (3% and 20 %) were injected through the applicator at a rate of 400 ml/h during the procedure. The pigs were followed up and they were euthanased on the 15th day. Impedance, current, power output, energy output, temperatures, diameters of thermal lesion, volume, sphericity ratio of thermal lesion were correlated among groups. Impedance at the end of the procedure (50.00 Ω±28.39 and 52.88 Ω±26.77, for groups A and B, respectively) was very similar to the starting impedance (50 Ω). In a median of 1 (range, 0–6) time per RF ablation procedure a reduction of 30 W from the selected power supply was observed during the RF ablation procedure linked to a slight increase of impedance. Volume and short diameter of thermal lesion were 21.28 cm3±11.78 and 2.85 cm±0.87 for group A, 87.51 cm3±25.20 and 4.31 cm±0.65 for group B. Continuous thermal between both electrodes were described with a global sphericity ratio of 1.91. One major complication (thermal injury to the stomach) was encountered in a case of cross-sectional necrosis of the targeted liver and attributed to heat diffusion after the procedure. This method has been shown to determine: (1) the relative control of impedance during the procedure; (2) ovoid and relatively large thermal lesions with less dependence upon closest vessels.
To test the viability of a new device to obtain hemostasis during laparoscopic partial nephrectom... more To test the viability of a new device to obtain hemostasis during laparoscopic partial nephrectomy (LPN) without vascular clamping. We performed a comparative experimental study between a new radiofrequency (RF)-assisted device consisting of a handheld instrument that simultaneously conducts coagulation and cutting tasks without hilar clamping vs a standard technique with hilar clamping. A porcine model was used (10 animals per group) with survival of 17 days. The estimated blood loss with the new device was significantly lower than with the standard technique (15.5±23.7 vs 79.4±76.3 mL). Although transection time was longer with the new device (10.7±13.7 vs 2.1±1.2 min), the total operative time was significantly shorter (35.3±13.7 vs 60.2±10.5 min). Evidence of localized urinary extravasation (urinoma) was identical in both groups (five cases). The group subjected to the new device, however, showed a significantly higher number of cases of leakage after conducting the methylene-blue test: eight (80%) cases vs only one (11%) with the standard technique. Necrosis depth was significantly greater with the new device (6.6±0.9 vs <1 mm). The experimental results suggest that the proposed RF-assisted device provides adequate hemostatic control during transection of the renal parenchyma without additional instruments or surgical maneuvers and could therefore be a valuable adjunct for LPN without vascular clamping. The device was unsuccessful in effectively sealing the collecting system.
The Cool-tip electrode is one of the most widely employed applicators in radiofrequency (RF) hepa... more The Cool-tip electrode is one of the most widely employed applicators in radiofrequency (RF) hepatic ablation. Previous research demonstrated that it is possible to enlarge coagulation volume when the single cooled electrode is associated with distant infusion of saline (hybrid applicator). The aim of this study was to compare the electrical-thermal behaviour of the Cool-tip electrode with that of the hybrid applicator. Forty-two RF ablations were performed on a total of 10 pigs: 22 with the Cool-tip electrode and 20 with the hybrid applicator (low infused saline volumetric flow rate of 6 mL/h at 2 mm distance). We compared both electrical performance (delivered power and number of roll-offs, i.e. sudden rises in impedance that interrupt the power delivery) and coagulation zone characteristics. In addition, we built a one-dimensional model to provide a basic physical explanation of the difference in performance between the different applicators. The experimental results showed that the number of roll-offs with the Cool-tip electrode was higher (24.3 ± 3.1 versus 6.7 ± 7.0). The hybrid applicator created larger coagulation volumes (19.7 ± 9.5 cm(3) versus 9.5 ± 5.8 cm(3)) with larger transverse diameters (2.5 ± 0.6 versus 1.9 ± 0.5 cm). The one-dimensional model confirmed the delay in the incidence of the first roll-off, but not the heterogeneity of the hybrid applicator's electrical performance in the experiments. The hybrid applicator produces fewer roll-off episodes than the Cool-tip electrode and creates larger coagulation volumes with larger transverse diameters.
We have examined the cellular distribution of both FGF-2 and FGFR1 immunoreactivity and their mRN... more We have examined the cellular distribution of both FGF-2 and FGFR1 immunoreactivity and their mRNAs throughout the normal adult rat brain in order to reconcile numerous disparate findings in the published literature. The results confirm a widespread distribution of FGF-2 and FGFR1 in the rat brain, and different regions express distinct patterns of FGF-2 and FGFR1 mRNA and protein: neuronal and non-neuronal cells show different subcellular distributions that vary according to the area where they are located. The intensity of the staining and hybridization also varies according to the loci examined and the cell type involved. Astrocytes contain the highest levels of FGF-2 and FGFR1 mRNAs, and characteristically, possess high levels of immunoreactive FGF-2 within the nucleus. Amongst non-neuronal cells, oligodendrocytes do not synthesize or contain significant levels of FGF-2 immunoreactivity however, they do express FGFR1 mRNA. In these cells, immunoreactive FGFR1 is mainly associated with the myelin sheaths of neuronal fibers. In ventricular systems, ependymal cells synthesize and contain immunoreactive FGFR1. In contrast, only cells lining the lateral wall of the IIIrd ventricle express FGF-2 mRNA. Subependymal cells contain high levels of both FGF-2 and FGFR1 immunoreactivity.Neurons express low levels of FGF-2 mRNA and immunoreactive FGF-2 is localized predominantly to the perikaryon. However, selected populations of neurons, such as CA2 field of the hippocampus, show high levels of FGF-2 mRNA, in which the nucleus is strongly immunopositive. Similarly, high levels of FGFR1 mRNA are localized to select populations of neurons (e.g. amygdala). FGFR1 immunoreactivity is mainly associated with myelinated fiber tracts (e.g. striatum), and some neurons show immunoreactivity in the perikaryon (e.g. hippocampus), the nucleus (e.g. mesencephalic trigeminal nucleus), or in axonal projections (e.g. hypothalamus). Remarkably, in many of the areas studied, FGF-2 and FGFR1 mRNA and/or their translated protein do not co-localize in neurons (e.g. neo-cortices) or even in the same regions of the brain (e.g. substantia nigra). In other instances, mRNAs for both FGF-2 and FGFR1 colocalize (e.g. supraoptic nucleus).The brain, in contrast to peripheral tissues, contains high levels of FGF-2 and actively expresses its gene under normal physiological conditions. The highly specific anatomical distribution of immunoreactive FGF-2 in neuronal and non-neuronal brain cells, supports the notion that it plays a multifunctional role in the CNS under normal physiology. By correlating the localization and the synthesis of FGF-2 and one of its high affinity receptors, FGFR1, in the CNS, it should be possible to obtain a better understanding of the roles of FGF-2 in normal and pathological conditions.
The single copy gene for human basic fibroblast growth factor (bFGF) has been shown to encode not... more The single copy gene for human basic fibroblast growth factor (bFGF) has been shown to encode not one but multiple proteins of 24, 23, 22 and 18 kD. Although bioactivities of the 18 kD protein are currently used to define bFGF gene function, it is not yet known if the three larger proteins have these same bioactivities or whether they will serve to define new bFGF gene functions. In this report we present a comparative study describing the de novo synthesis, transport, processing and intracellular location of individual bFGF isoforms. Data from cDNA mutagenesis and COS cell expression experiments show that individual isoforms are differentially localized to either the cell surface or to the nucleus. The 24, 23 and 22 kD proteins (CUG-mediated initiation) exclusively localize in the nucleus while the 18 kD protein (AUG-mediated initiation) is preferentially exported onto the cell surface, but is not released into the surrounding culture medium. Specific CUG or AUG translation initiation codons are necessary and sufficient for the synthesis of each isoform examined and thereby, indirectly, mediate differential localization. Since bFGF does not contain the characteristic signals predicted for cell surface or nuclear targeting, our continuing studies will either unmask its functionally equivalent domain(s) or will identify the requisite participation of yet unknown cellular components.
In the central nervous system (CNS), nerve regeneration after traumatic injury fails. The formati... more In the central nervous system (CNS), nerve regeneration after traumatic injury fails. The formation of a dense fibrous scar is thought to restrict in part the growth of axonal projections, providing one of the many reasons that complete lesions of neural pathways in the adult mammalian CNS are rarely followed by significant functional recovery. In order to determine which mechanisms mediate scar formation in the CNS and to investigate whether they can be modulated in vivo, we have attempted to define the potential role of trophic factors. Our previous studies have shown the focal elevation of transforming growth factor β1 (TGFβ1) expression in lesioned CNS tissue. In the studies described here, we demonstrate that TGFβ1 participates in the scarring response in the rat brain. First, the elevated protein levels of TGFβ1 are localized to specific populations of injury-responsive cells in the traumatized CNS. Furthermore, the injection of TGFβ1 into the brains of injured rats causes a dramatic increase in the scarring response. Conversely, when neutralizing TGFβ1 antibodies are administered, the deposition of fibrous scar tissue and the formation of a limiting glial membrane that borders the lesion is significantly attenuated, thus establishing a role for the endogenous growth factor in regulation of the non-glial component of the scar. In implicating TGFβ1 in the scarring response in the CNS, the potential use for TGFβ1 antagonists as inhibitors of scar formation in the injured mammalian CNS is self-evident.
The expression of basic FGF mRNA, while virtually absent in peripheral tissues, appears to be con... more The expression of basic FGF mRNA, while virtually absent in peripheral tissues, appears to be constitutively expressed in the central nervous system. As such, while it is difficult to detect any mRNA encoding basic FGF in the heart, lung, kidneys, ovaries, liver, or pituitary of rats, the levels are easily detected in brain. A regional analysis of the expression of basic FGF mRNA in brain reveals that it is widely distributed in the cortex (frontal, parietal, and occipital), the hippocampus, hypothalamus, and pons. Only a few loci of basic FGF synthesis are detected by in situ hybridization and include layers 2 and 6 of the medial (cingulate) cortex, the indusium griseum, fasciola cinereum, and field CA2 of the hippocampus. The identification of specific cell populations in the brain, and particularly in the hippocampus, that synthesize basic FGF supports the notion that this potent neurotrophic factor is involved in normal CNS function and that the presence (or absence) of its expression may be linked to the pathogenesis of the neurogenerative diseases characterizing these various loci. The significance of these findings with respect to the regulation of basic FGF expression in peripheral tissue and the central nervous system is discussed.
Bipolar radiofrequency (RF) ablation, especially with perfusion of saline, has been shown to incr... more Bipolar radiofrequency (RF) ablation, especially with perfusion of saline, has been shown to increase volume over monopolar conventional methods. The aims of this study are to study whether this method is linked to too flattened thermal lesions and premature rise of impedance and to elucidate some safety concerns. Eighteen RF ablations were performed using a 1.8-mm-diameter bipolar applicator in the liver of nine healthy pigs through laparotomy with or without temporary vascular occlusion [the Pringle maneuver (PGM)]: group A (n=9), without PGM; group B (n=9), with PGM. Hypertonic saline solutions (3% and 20 %) were injected through the applicator at a rate of 400 ml/h during the procedure. The pigs were followed up and they were euthanased on the 15th day. Impedance, current, power output, energy output, temperatures, diameters of thermal lesion, volume, sphericity ratio of thermal lesion were correlated among groups. Impedance at the end of the procedure (50.00 Ω±28.39 and 52.88 Ω±26.77, for groups A and B, respectively) was very similar to the starting impedance (50 Ω). In a median of 1 (range, 0–6) time per RF ablation procedure a reduction of 30 W from the selected power supply was observed during the RF ablation procedure linked to a slight increase of impedance. Volume and short diameter of thermal lesion were 21.28 cm3±11.78 and 2.85 cm±0.87 for group A, 87.51 cm3±25.20 and 4.31 cm±0.65 for group B. Continuous thermal between both electrodes were described with a global sphericity ratio of 1.91. One major complication (thermal injury to the stomach) was encountered in a case of cross-sectional necrosis of the targeted liver and attributed to heat diffusion after the procedure. This method has been shown to determine: (1) the relative control of impedance during the procedure; (2) ovoid and relatively large thermal lesions with less dependence upon closest vessels.
To test the viability of a new device to obtain hemostasis during laparoscopic partial nephrectom... more To test the viability of a new device to obtain hemostasis during laparoscopic partial nephrectomy (LPN) without vascular clamping. We performed a comparative experimental study between a new radiofrequency (RF)-assisted device consisting of a handheld instrument that simultaneously conducts coagulation and cutting tasks without hilar clamping vs a standard technique with hilar clamping. A porcine model was used (10 animals per group) with survival of 17 days. The estimated blood loss with the new device was significantly lower than with the standard technique (15.5±23.7 vs 79.4±76.3 mL). Although transection time was longer with the new device (10.7±13.7 vs 2.1±1.2 min), the total operative time was significantly shorter (35.3±13.7 vs 60.2±10.5 min). Evidence of localized urinary extravasation (urinoma) was identical in both groups (five cases). The group subjected to the new device, however, showed a significantly higher number of cases of leakage after conducting the methylene-blue test: eight (80%) cases vs only one (11%) with the standard technique. Necrosis depth was significantly greater with the new device (6.6±0.9 vs <1 mm). The experimental results suggest that the proposed RF-assisted device provides adequate hemostatic control during transection of the renal parenchyma without additional instruments or surgical maneuvers and could therefore be a valuable adjunct for LPN without vascular clamping. The device was unsuccessful in effectively sealing the collecting system.
The Cool-tip electrode is one of the most widely employed applicators in radiofrequency (RF) hepa... more The Cool-tip electrode is one of the most widely employed applicators in radiofrequency (RF) hepatic ablation. Previous research demonstrated that it is possible to enlarge coagulation volume when the single cooled electrode is associated with distant infusion of saline (hybrid applicator). The aim of this study was to compare the electrical-thermal behaviour of the Cool-tip electrode with that of the hybrid applicator. Forty-two RF ablations were performed on a total of 10 pigs: 22 with the Cool-tip electrode and 20 with the hybrid applicator (low infused saline volumetric flow rate of 6 mL/h at 2 mm distance). We compared both electrical performance (delivered power and number of roll-offs, i.e. sudden rises in impedance that interrupt the power delivery) and coagulation zone characteristics. In addition, we built a one-dimensional model to provide a basic physical explanation of the difference in performance between the different applicators. The experimental results showed that the number of roll-offs with the Cool-tip electrode was higher (24.3 ± 3.1 versus 6.7 ± 7.0). The hybrid applicator created larger coagulation volumes (19.7 ± 9.5 cm(3) versus 9.5 ± 5.8 cm(3)) with larger transverse diameters (2.5 ± 0.6 versus 1.9 ± 0.5 cm). The one-dimensional model confirmed the delay in the incidence of the first roll-off, but not the heterogeneity of the hybrid applicator's electrical performance in the experiments. The hybrid applicator produces fewer roll-off episodes than the Cool-tip electrode and creates larger coagulation volumes with larger transverse diameters.
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Papers by Ana Gonzalez