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B T Stokes

Publications and source records attributed to B T Stokes.

At least 55 records · Page 3Linked to original sources
PubMed1994

The effects of gonadotropin on the phosphatidylinositol pathway in the primate corpus luteum.

The current study was designed to investigate the effects of gonadotropin on basal and prostaglandin (PG) F2 alpha-induced activity of the phosphatidylinositol pathway in corpora lutea (CL) of rhesus monkeys. Luteal progesterone production in vitro was significantly stimulated (P < 0.05) by human chorionic gonadotropin (hCG). Neither basal nor PGF2 alpha-induced phosphatidylinositol 4,5-bisphosphate hydrolysis was significantly influenced by hCG in CL of various ages (P > 0.10). Gonadotropin did induce a slight, yet sustained, increase (P < 0.05) in [Ca2+]i in approximately 70% of luteal cells. The maximal increase in [Ca2+]i in response to hCG (approximately 100 nM) was about one-tenth that induced by PGF2 alpha (approximately 1000 nM). hCG treatment did not alter (P > 0.10) the increase in [Ca2+]i induced by PGF2 alpha Treatment-induced changes in [Ca2+]i did not differ between small (17-21 microns) and large (23-28 microns) luteal cells. Therefore, luteolytic agents are more potent activators of the phosphatidylinositol pathway than luteotropins. This is consistent with the hypothesis that the phosphatidylinositol pathway is involved in primate luteal regression. The inability of hCG to acutely alter the responsiveness of this pathway to PGF2 alpha suggests that CG may rescue the CL of early pregnancy via a mechanism other than direct inhibition of the luteolytic actions of PGF2 alpha.

Animals↗
PubMed1994

Procaine, lidocaine, and hypothermia inhibit calcium paradox in glial cells.

The purpose of this study was to determine the effects of local anesthetics (lidocaine and procaine) and hypothermic perfusion on Na+/Ca2+ exchanger activity during reperfusion-induced calcium paradox in vitro. Hypothetically, this work provides a mechanism for reperfusion paradox injury in vitro that may relate to a variety of ischemic insults in vivo. Intracellular calcium ([Ca2+]i) in individual cells was measured using a fura-2 photometry system during protocols previously shown to dramatically and persistently increase [Ca2+]i in a manner that replicates certain phases of reperfusion injury in vivo. Our results suggest that short (< 5.0 min) exposure to hypocalcia prompts subsequent, potentially lethal, sodium-dependent calcium entry via the reverse phase of the Na+/Ca2+ exchanger. The local anesthetics lidocaine or procaine are able to block [Ca2+]i increases when added at the time of reperfusion. Furthermore, since [Ca2+]i is normally increased when local anesthetics are added by themselves, the results suggest that local anesthetics promote the forward mode of Na+/Ca2+ exchange (i.e., Na-dependent calcium removal) in addition to reverse Na+/Ca2+ exchanger blockade. Low temperature (18 degrees C) perfusion or reperfusion also affects exchanger dynamics and attenuates calcium entry during such protocols. These experiments further implicate the Na+/Ca2+ exchanger in reperfusion-mediated cell injury and provide potential ways to therapeutically modify such phenomena in vivo.

Analysis of Variance↗
PubMed1993

Intracellular calcium dynamics and cerebral injury: modeling various insults in vitro.

The magnitude and time course of intracellular [Ca2+]i alterations were studied after excitatory amino acid challenge (EAA) or chemical energy depletion in mature spinal cultures. While either cytotoxic event led to prompt increases in [Ca2+]i, the pattern of these changes before and after exposure to the toxin was different. EAA [Ca2+]i changes seem primarily dependent on surface membrane alterations from which the cells rapidly recover while energy depletion effects release of [Ca2+]i from intracellular stores and produces a lasting compromise in the ability of these neurons in culture to recover from the initial insult.

Brain Injuries↗
PubMed1993

Mechanisms through which gangliosides inhibit PDGF-stimulated mitogenesis in intact Swiss 3T3 cells: receptor tyrosine phosphorylation, intracellular calcium, and receptor binding.

Several potential mechanisms through which gangliosides could modulate PDGF-stimulated events in Swiss 3T3 cells were studied using intact cells. Of the gangliosides studied, at low micromolar concentrations GM2 was the only one that inhibited PDGF-stimulated DNA synthesis, but GT1b was the most potent between 25 and 100 microM; GM1 was generally the least effective, and GD1a and GM3 had intermediate effects. All gangliosides tested inhibited the PDGF-stimulated increases in free intracellular calcium concentrations ([Ca2+]i) with the rank order of potency being GM1 > or = GT1b > GM2 > GM3. PDGF stimulated phosphorylation on tyrosine of a protein with apparent M(r) = 170 kDa which was immunoprecipitated by an anti-PDGF receptor (beta) antibody, indicating that it is a PDGF receptor. Preincubating the cells with specific gangliosides inhibited tyrosine phosphorylation of this protein in a dose-responsive fashion with the following rank order of potency GD1a = GT1b > GM1 > GM2 > GM3. Autoradiography showed that this was due to a decrease in the proportion of cells synthesizing DNA, and a time study showed that ganglioside did not delay entry of the cells into S phase. These effects were not due to gangliosides interfering with PDGF binding to its receptor because results of competitive binding studies showed that none of the gangliosides studied had an effect on either receptor number or affinity, and did not bind to PDGF in solution. These results show that gangliosides affect several specific components of the complex responses to PDGF in intact cells. The relative effectiveness of individual gangliosides, however, varied among the different cellular and molecular responses. This is interpreted to mean that specific gangliosides modulate to different degrees several molecular mechanisms which converge on the common biological response of mitogenesis, and suggests that gangliosides as a family of molecules may function as coordinators of different molecular events involved in complex cellular processes.

3T3 Cells↗
PubMed1993

Myoelectric evoked potentials versus locomotor recovery in chronic spinal cord injured rats.

The purpose of this study was to determine the utility of descending evoked potentials in evaluating functional recovery in rats after spinal cord contusion injury. Rats received thoracic contusions at T9 using a controlled-displacement impactor. They were evaluated for 5 weeks postinjury using auditory startle responses (ASR) while alert, or by cerebellar motor evoked potentials (CMEP) while anesthetized. ASR and CMEP were recorded electromyographically from forelimb and hindlimb muscles. Open field locomotor performance was also assessed and recovered to almost normal levels by 3 weeks postinjury. Histologic analysis of the injury site indicated that the contusions destroyed approximately 70% of the cross-sectional area of the cord. Although the remaining 30% was sufficient to preserve nearly normal locomotor behavior, ASR and CMEP amplitudes in hindlimb flexors and extensors were reduced by 90% or more after injury and showed virtually no recovery. Significant ASR and CMEP responses were present in the cutaneous trunk muscles of the lower torso after injury. These muscles are innervated via peripheral nerves originating at cord levels above the injury. Multi-wave field potentials normally recorded from the dorsal cord surface in response to cerebellar stimulation were absent in injured rats, suggesting minimal if any activation of segmental neurons via the pathways normally mediating CMEP. The tracts mediating ASR and CMEP thus appear to be highly sensitive to mild spinal cord trauma but are evidently not essential for support or walking.

Acoustic Stimulation↗
PubMed1993

Differential expression of MHC class II antigen in the contused rat spinal cord.

Following contusion injury to the dorsal surface of thoracic rat spinal cord, major histocompatibility complex (MHC) class II (Ia) antigen expression by microglia was evaluated throughout the developing lesion. Past investigations of various central nervous system (CNS) lesions have examined short-term or acute sequelae of post-traumatic Ia expression. This report demonstrates that in animals allowed to recover for 18 (sub-chronic) and 45 (chronic) days post-injury, MHC class II antigen is expressed differently at rostral and caudal extents of the lesion as compared with the lesion's epicenter. Following contusion injury to the thoracic spinal cord, sub-chronically injured animals demonstrated Ia-positive microglial staining throughout the white matter rostral and caudal to the epicenter of the lesion, whereas Ia-positive microglia and/or perivascular cells are localized within the gray matter adjacent to it. MHC class II immunoreactivity is down-regulated on microglia at chronic survival times but clusters of Ia-positive macrophages are prominent in regions of maximal degeneration at the epicenter of the lesion. Our findings support the theory that two distinct populations of macrophages participate in resolving traumatic injury. One population is the parenchymal CNS microglia and the other is presumably exudate macrophages derived from the blood. Furthermore, the immunocompetence of these cells as measured by MHC expression may be differentially regulated. This hypothesis is based on differences in Ia-positive staining observed between microglia and macrophages over time concomitant with differences in the spatial distribution of these cell types.

Animals↗
PubMed1992

Gangliosides inhibit platelet-derived growth-factor-stimulated increases in intracellular calcium in Swiss 3T3 cells.

Individual Swiss 3T3 cells stimulated by platelet-derived growth factor delivered by means of a picopump device respond with a brisk, large, and sustained increase in intracellular calcium concentration ([Ca2+]i). Preincubation of cells with either GM1 or GT1b gangliosides inhibited the proportion of responding cells and caused a dose-related diminution in the magnitude of the increase in [Ca2+]i. This effect of ganglioside is probably part of the mechanism through which gangliosides exert their biological effects, including inhibition of platelet-derived growth-factor-induced mitogenesis.

3T3 Cells↗
PubMed1992

Reperfusion paradox: a novel mode of glial cell injury.

We have attempted to reconstruct in vitro the events that may occur in vivo during reperfusion injury after ischemia in the central nervous system. The phenomenon is induced by previous exposure to low calcium solutions ("calcium paradox") before the reperfusion episode. Intracellular calcium alterations during reperfusion of human astrocytoma U1242MG cells have been investigated with microspectrofluorimetry using the calcium-sensitive dye fura-2. Cells were perfused in calcium-free buffer solution for 30 min and then re-exposed to the control buffer solution (1.5 mM CaCl2). [Ca2+]i increased up to 3.5 times control levels during the reperfusion period. The mechanism of the increase was also investigated. Addition of TTX (2 microM) or choline chloride sodium substitution during perfusion with low calcium prevented the [Ca2+]i increase during reperfusion. Reperfusion increases in [Ca2+]i were exacerbated by low potassium in the perfusion medium, but unaltered by the calcium channel blockers cadmium (100 microM) and nickel (100 microM). In a similar manner, flunarizine (10 microM) and cadmium (100 microM) were unable to modify reperfusion [Ca2+]i alterations. Low sodium in the reperfusion medium produced significant increases in [Ca2+]i if preceded by low potassium and calcium perfusion. The viability of cells after 24 h of incubation after the insult produced by exposure to Ca(2+)-free media for 30 min was also investigated. Compared with control groups, the groups treated with Ca(2+)-free media for 30 min had a decreased number of surviving cells and morphological alterations indicative of cell pathology. The relative number of cytotoxic cells was increased by maneuvers (low potassium perfusion) that presumably blocked the Na/KATPase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Fetal grafts alter chronic behavioral outcome after contusion damage to the adult rat spinal cord.

In the present experiments, we have examined the capacity of intraspinal transplants to effect alterations in certain locomotor behaviors after spinal contusion injuries. An electromechanical impactor that was sensitive to tissue biomechanical characteristics was used to produce rapid (20 ms) compression injuries to the thoracic spinal cord (T8). Suspensions of fetal spinal tissue (14-day) were placed at 10 days postinjury into the intraspinal cavity created by these reproducible spinal injuries. In the pre- and postinjury period, a number of general and sensitive motor behaviors were used to characterize the immediate and long-term progress of hindlimb behavioral recovery over an extended period of time (73 days). Our data reveal that a lasting alteration in some motor behaviors can be achieved by suspension grafts. While little improvement in some generalized motor tasks (inclined plane analysis, grid walking) takes place, fetal transplants precipitate a rapid and enduring change in certain motivated fine motor behaviors (gait analysis). The base of support and stride length of the hindlimbs were improved by 7 days post-transplantation and the effect was stable over time. The angle of rotation was, however, not altered. The lasting effect in two gait parameters noted was accompanied by the presence of well-developed spinal grafts that often fused with the host spinal parenchyma. These results provide the first documentation of an influence of fetal transplants on motivated locomotor capacity in a well-characterized spinal injury model that mimics lesions seen in the contused adult human spinal cord.

Animals↗
PubMed1992

Fetal cell grafts into resection and contusion/compression injuries of the rat and cat spinal cord.

This article reviews recent findings concerning the feasibility, basic neurobiology, and potential functional benefits of fetal CNS tissue grafts into acute and chronic lesions of the adult spinal cord. In the rat, neuro-anatomical observations suggest that transplants into resection cavities establish neuritic projections that could functionally reunite separated rostral and caudal segments of the host spinal cord. Furthermore, some complementary electrophysiological evidence has been obtained for synaptic connectivity between host and graft neurons. In these studies, extracellular single-unit activity was evoked in fetal spinal cord (FSC) transplants by stimulating host dorsal roots that had been juxtaposed to donor tissue at the time of transplantation. In other investigations, we examined whether grafts could also establish axonal projections to appropriate areas of gray matter in the chronically injured spinal cord. For this purpose, fetal serotoninergic (5-HT) neurons were injected caudal to complete spinal cord transections that had been made 1-3 months earlier. Immunocytochemistry revealed that these cells projected their axons into gray matter regions normally innervated by bulbospinal 5-HT neurons. To investigate transplantation in a more clinically relevant lesion model, a third group of experiments involved injection of dissociated cell suspensions into acute [less than 24 h postinjury (p.i.)]), subchronic (7-10 days p.i), and chronic (greater than or equal to one month, p.i.) contusion lesions. Such grafts routinely filled areas that otherwise would have been regions of cavitation extending rostral-caudal distances of approximately 7 mm. FSC transplants in such injuries also appeared to influence some aspects of motoneuron excitability and hindlimb locomotion. More recent studies of the cat spinal cord have extended these findings in the rat by showing long-term survival (greater than 2 years) of fetal CNS allografts in recipients with either subtotal transection or compression lesions. Preliminary studies of connectivity have also shown host-graft projection patterns similar to those seen in the rat. Behavioral analyses are currently underway to examine the effects of fetal grafts in cats with chronic postcompression lesions. These observations in the rat and cat are discussed in the general context of basic biological and clinical issues relevant to the long-term objective of promoting functional improvement in the damaged spinal cord.

Animals↗
PubMed1992

An electromechanical spinal injury technique with dynamic sensitivity.

Over the past decade, our laboratory has attempted to create a simple, accurate device that could be used to produce reliable and quantifiable spinal cord injuries in the rodent. We report here on our latest of several modifications of a spinal cord impactor that has allowed us to meet these design criteria. The impactor uses the dynamic capacity of an electromagnetic driver (Ling shaker) and a unique pattern generator to briefly compress the dorsal surface of the spinal cord at velocities that may mimic compression injuries seen in the human. Calibrated, independent transducer systems provide open-loop output of the precise movement (displacement) of the impactor probe and the force necessary to achieve a given displacement. Touch sensitivity is accomplished by vibrating the probe slightly as it approaches the dural surface. This also allows a known biomechanical starting point. This combination of improvements in sensitivity and ability to measure all components of the dynamic compression has allowed us to determine detailed biomechanical descriptors of these impact injuries with low coefficients of variation. Furthermore, such descriptors correlate highly with histopathologic and behavioral outcome measures in animal populations with a variety of injury severities.

Animals↗
PubMed1992

Activation of the phosphatidylinositol pathway in the primate corpus luteum by prostaglandin F2 alpha.

The current study was designed to investigate the ability of prostaglandin F2 alpha (PGF2 alpha) to activate a second messenger system (phosphatidylinositol pathway) in corpora lutea (CL) of rhesus monkeys. Activation of this pathway was assessed by monitoring the hydrolysis of phosphatidylinositol to inositol phosphates. Since inositol triphosphate mobilizes intracellular Ca2+, intracellular free calcium concentrations ([Ca2+]i) were also assessed in individual cells by fura-2 fluorescence photometry. These responses to PGF2 alpha were measured in luteal cells collected from nonpregnant rhesus monkeys. CL were collected during the early (days 4-5 after estimated LH surge; n = 4), mid (days 8-9; n = 4), and late (days 13-14; n = 5) luteal phase and 1 day after in vivo hCG treatment (15 IU/dose, morning and evening), which began during the midluteal phase (n = 5). PGF2 alpha significantly increased the accumulation of inositol phosphates in all groups (P less than 0.05), except the midluteal phase (P = 0.07). The luteal sensitivity to PGF2 alpha, judged by phosphatidylinositol hydrolysis, was low in the early to midluteal phase compared to that in the late luteal phase and after in vivo hCG treatment. PGF2 alpha also caused a rapid, yet transient, increase in [Ca2+]i in a large proportion of primate luteal cells. The proportion of luteal cells that responded to PGF2 alpha with an increase in [Ca2+]i was smaller (P less than 0.05) in CL collected during the early luteal phase than in the other groups. Luteal progesterone production was inhibited by PGF2 alpha in CL collected after in vivo hCG. CL treated in vivo with hCG also displayed in vitro the largest increases in phosphatidylinositol hydrolysis and [Ca2+]i in response to PGF2 alpha. Therefore, this study demonstrates that PGF2 alpha is a potent activator of the phosphatidylinositol pathway in the primate CL. This activation is augmented as the luteal phase progresses and is influenced by in vivo hCG treatment. This study also provides evidence that the inhibitory effects of PGF2 alpha on progesterone production are associated with the activation of the phosphatidylinositol pathway.

Animals↗
PubMed1992

Experimental models for spinal cord injury research: physical and physiological considerations.

This paper describes historical and current experimental models used to develop our current understanding of the biomechanics and pathophysiology of traumatic spinal cord injury; the advantages and limitations of current experimental models; considerations for selecting an appropriate injury model based on experimental objectives; and key physiological factors in the spinal cord injury response that may interact with the injury response and alter the outcome. All of the above must be considered in the development and selection of an appropriate experimental injury model that meets specific needs. Various experimental models have been developed to study spinal cord injury and the pathophysiological and physical mechanisms responsible for tissue damage and loss of function. Such modeling may involve inherently different biomechanical variables with alternative outcomes and purposes. There is not, therefore, a single "ideal" experimental injury model just as there is no "stereotypical" clinical spinal cord injury. Instead, the goals and objectives of the research dictate specific requirements on the model. In all cases, however, both physical and physiological aspects of the model should be considered, and measured if possible, to ensure interlaboratory comparability and possible clinical relevance. Also, experimental techniques, especially anesthesia, and surgical procedures, should be carefully reviewed for interactions with the injury response or potential therapeutic interventions to ensure validity of interpretation. It is hoped that data correlating physical spinal cord injury parameters with functional outcome will ultimately be combined with data on vertebral injury and spinal failure mechanics to further our understanding of clinical injury. Such approaches should lead to interventions that reduce the incidence and severity of traumatic human spinal cord injury.

Animals↗
PubMed1992

Neural tissue transplantation and CNS trauma: anatomical and functional repair of the injured spinal cord.

Neural tissue transplantation has become recognized widely as a powerful experimental tool for studying structure-function relationships, development, plasticity, and capacities for regeneration in the adult CNS. In addition, this area of investigation has generated considerable interest in approaches that might be applicable to a variety of catastrophic neurological disorders. In this regard, attention has been given to neural tissue grafting as a potential therapeutic strategy in various forms of neurodegenerative disease. More recently, however, other investigations have begun to focus on the possible application of peripheral and central neural tissue transplants for promoting repair in forms of CNS trauma. This review highlights various neural transplantation approaches that have been explored primarily in the context of injury to the adult CNS, with emphasis on spinal cord injury. An overview is presented of the evolution of this area of research in terms of emerging biological perspectives, technological advances, and experimental modelling. Discussion centers on progress that has been made and a variety of theoretical and practical issues that remain to be resolved.

Animals↗
PubMed1991

Energy depletion in culture. Adenine nucleotides are altered as in vivo.

Modeling of ischemic phenomena in vitro has been hindered by the inability to create specific alterations in the variables of interest over a defined time-frame. In particular, changes in the adenine nucleotide pool have been quite difficult to mimic because of the putative low metabolic rate in culture and the long times necessary to achieve even partial chemical energy depletion. Here we present evidence for a rapid method of producing a profound chemical energy depletion with the combination of a NADH dehydrogenase inhibitor (amytal) and a mitochondrial proton ionophore (CCCP). Treatment with our protocol in enriched spinal cultures results in a 40% decrease in ATP within 2 min and a fall to one-third of control values by 15 min. The overall pool size of the total adenine nucleotides is decreased 46% by 15 min and does not completely recover after 5 min of reenergization. The ATP/ADP ratio declines to one-third of control values during deenergization and returns to control values after 5 min in control buffer. Such a loss of the total adenylate pool closely mimics that seen in vivo during ischemia and provides an in vitro model system in which the effects of the combination of this means of cellular injury with others (e.g., excitotoxins) may be examined.

Adenine Nucleotides↗
PubMed1991

Oxygen transport in intraspinal fetal grafts: graft-host relations.

In the present experiments, we determined tissue oxygen tension (PtO2) levels within the injured spinal cords of adult rats following transplantation of fetal spinal cord tissue. Partial resection cavities were made at L1-L2 levels after which whole pieces of 14-day donor tissue were placed into the cavities. Analysis of recordings obtained from graft tissues at 1 and 2 months after transplantation revealed low PtO2 values in many cases. Even the more extensively developed transplants at 3 months continued to show mean PtO2 levels lower than those taken from normal, mature spinal cord tissue. Measurements from host tissue, adjacent to a lesion in which no graft was introduced, showed normal or elevated PtO2 levels. In contrast, where extensive host and graft integration had occurred, the PtO2 levels of adjacent host resembled those obtained within the transplants. On the other hand, in cases of poor host-graft integration, characterized by either cellular or fibrotic graft-host interfaces or large cysts, the PtO2 tensions exceeded normal levels. Therefore, the present results show that when fetal grafts are placed acutely into an aspiration cavity within the adult spinal cord, the transplants quickly establish an oxygen microenvironment resembling that found during normal fetal development. Oxygen transport is therefore a regulated variable in the graft neuropil as it is in the normally developing spinal cord. Furthermore, in the presence of closely approximated fetal transplants, adjacent host tissue assumes tissue oxygen levels that mimic those in the graft. This "inductive" effect gradually diminishes as development proceeds and may be the hallmark of successful graft-host integration.

Animals↗
PubMed1991

Determination of Fura-2 dissociation constants following adjustment of the apparent Ca-EGTA association constant for temperature and ionic strength.

The accurate calibration of Fura-2 fluorescence in living cells is dependent upon the apparent dissociation constant (Kd) of Fura-2 for Ca2+. If Ca-EGTA calibration buffers are used to construct an in vitro calibration curve, then the calculated value of the apparent Ca-EGTA association constant (K'CaEGTA) will have an important influence on the Kd of Fura-2 and thus the calculated free [Ca2+] in cells. In order to simulate experimental conditions, the individual proton and Ca2+ association constants for EGTA in these experiments were adjusted for both ionic strength and temperature using a semi-empirical form of the Debye-Huckel limiting law and the Van't Hoff isochore, respectively, as described by Harrison and Bers. The modified individual binding constants were then employed in the calculation of K'CaEGTA using the SPECS computer program of Fabatio. At pH = 7.05, ionic strength = 0.15 M, temp = 20 degrees C, K'CaEGTA = 3.232 x 10(6) M-1; at pH = 6.84, temp = 36 degrees C, K'CaEGTA = 1.652 x 10(6) M-1. These values differed substantially from those obtained with unadjusted individual association constants. Calibration buffers of varying [Ca2+] were prepared using the corrected values of K'CaEGTA, and Fura-2 fluorescence ratios were measured during superfusion of these buffers in the experimental chamber at both 20 degrees C and 37 degrees C. The Kd of Fura-2 for Ca2+ was determined to be 236 nM at 20 degrees C and 285 nM at 37 degrees C, utilizing the value of K'CaEGTA adjusted by the method of Harrison and Bers.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗