Biomedical subjects
F R Sharp
Publications and source records attributed to F R Sharp.
Ascorbate neurotoxicity in cortical cell culture.
Ascorbate (vitamin C) is believed to act as a neuromodulator that facilitates the release of neurotransmitters and inhibits neurotransmitter binding to receptors, including dopamine and N-methyl-D-aspartate receptors. Extracellular levels of ascorbate are known to reach the low millimolar range after ischemic brain injury. This study shows that treatment of cultured cortical neurons with micromolar to low millimolar ascorbate first inhibits total protein synthesis and then results in late neuronal death. Astrocytes are much less vulnerable to ascorbate than neurons. Ascorbate may exacerbate neuronal and glial damage after brain ischemia, and it may play a pathological role in other neurological diseases.
Zinc toxicity and induction of the 72 kD heat shock protein in primary astrocyte culture.
Zinc is a potent inducer of the 72 kD heat shock protein (HSP72). In brain, pathological conditions such as ischemia and seizures increase extracellular zinc. The present study examines the effect of zinc on HSP72 expression in rat primary cortical astrocyte culture. Astrocytes were grown to confluence and exposed to zinc chloride in CO2-equilibrated Earle's buffered salt solution. Expression of HSP72 was examined using immunocytochemistry. HSP72 was induced with zinc concentrations of 5 to 100 microM after 4 h exposures, or 200 to 300 microM after 15 min exposures. At the lower concentrations expression occurred in small clusters of contiguous cells. At concentrations high enough to cause cell death, HSP72-positive astrocytes formed a continuous margin around patches of dead cells. These patterns of HSP72 expression are similar to the patterns seen after cerebral ischemia in vivo. Exposure to zinc at 100 microM for 4 h or 400 microM for 15 min caused greater than 90% cell death. Increases in extracellular zinc may contribute to HSP72 induction and astrocyte death under ischemia and other pathological conditions in brain.
Haloperidol prevents induction of the hsp70 heat shock gene in neurons injured by phencyclidine (PCP), MK801, and ketamine.
The non-competitive NMDA receptor antagonists, PCP (phencyclidine), MK801, and ketamine produce psychosis in humans and abnormal vacuoles in posterior cingulate and retrosplenial rat cortical neurons. We show that PCP (> or = 5 mg/kg), MK801 (> or = 0.1 mg/kg), and ketamine (> 20 mg/kg) induce hsp70 mRNA and HSP70 heat shock protein in these vacuolated, injured neurons, and PCP also induces hsp70 in injured neocortical, piriform, and amygdala neurons. The PCP, MK801, and ketamine drug induced injury occurs in 30 day and older rats, but not in 0-20 day old rats, and is prevented by prior administration of the antipsychotic drugs haloperidol and rimcazole. Since haloperidol and rimcazole block dopamine and sigma receptors, and since M1 muscarinic cholinergic receptor antagonists also prevent the injury produced by PCP, MK801, and ketamine, future studies will be needed to determine whether dopamine, sigma, M1, or other receptors mediate the injury.
Sensory stimulation induces local cerebral glycogenolysis: demonstration by autoradiography.
Brain glycogen stores are localized primarily to glia and undergo continuous utilization and resynthesis. To study the function of glycogen under normal conditions in brain, we developed an autoradiographic method of demonstrating local-glycogen utilization in the awake rat. The method employs labeling of brain glycogen with 14C(3,4)glucose, in situ microwave fixation of brain metabolism, and anhydrous tissue preparation. With this technique, tactile stimulation of the rat face and vibrissae was found to accelerate the utilization of labeled glycogen in brain regions known to receive sensory input from face and vibrissae: the contralateral somatosensory cortex and the ipsilateral trigeminal, sensory and motor nuclei. These findings demonstrate a link between neuronal activity and local glycogen utilization in mammalian brain and suggest that, like other tissues, brain may respond to sudden increases in energy demand in part by rapid glycolytic metabolism of glycogen. As cerebral glycogen is restricted primarily to glia, these observations also support a close coupling of glial energy metabolism with neuronal activity.
C-fos induction occurs in cultured cortical neurons and astrocytes via multiple signaling pathways.
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N-methyl-D-aspartate antagonists block fos-like protein expression induced via multiple signaling pathways in cultured cortical neurons.
c-fos mRNA and Fos-like protein(s) (FLP) are induced in cultured cortical neurons by glutamate, high K+, phorbol ester, basic fibroblast growth factor, Zn2+, and vasoactive intestinal peptide. Glutamate induction of c-fos mRNA and FLP is blocked by noncompetitive N-methyl-D-aspartate (NMDA) antagonist, MK-801, and competitive NMDA antagonists, 4-(3-phosphonopropyl)piperazin-2-carboxylic acid and 2-amino-7-phosphonoheptanoate. These antagonists partially block high K(+)-, phorbol ester-, Zn(2+)-, and VIP-induced c-fos mRNA expression, but have no effect on bFGF-induced c-fos mRNA expression. However, both competitive and noncompetitive NMDA antagonists completely block FLP induction by all of these agents without affecting total protein synthesis. Therefore, these NMDA antagonists block FLP translation, without blocking c-fos transcription. It is hypothesized that NMDA receptor activation is required for translation of c-fos mRNA in cortical neurons after stimulation of multiple intracellular signaling pathways. It is possible that NMDA antagonists prevent cortical plasticity by blocking induction of the Fos protein that would normally be induced by neurotrophic factors, neurotransmitters, and neuromodulators.
Long experimental durations are required for double label [14C]- and [3H]2-deoxyglucose autoradiographic methods.
Double-label 2-deoxyglucose (2-DG) studies using sequential [14C]- and [3H]2-DG injections demonstrate increased [14C]2-DG uptake during the first and second stimulation periods. To understand why this occurs, the rat mystacial vibrissae were stimulated at various times following [14C]2-DG injection. Local cerebral glucose utilization (LCGU) increased when whisker stimulation was performed at 0-90 min following [14C]2-DG injection. LCGU did not increase when whisker stimulation was performed at 90-150 min following [14C]2-DG injection. To minimize contamination of the two tracers in double label 2-DG mapping studies, the time between [14C]- and [3H]2-DG administration should be increased to 90 min.
MK-801 and ketamine induce heat shock protein HSP72 in injured neurons in posterior cingulate and retrosplenial cortex.
MK-801 and ketamine are noncompetitive N-methyl-D-aspartate (NMDA) receptor blockers that decrease brain injury in animal models of focal and global ischemia. Recent reports, however, suggested that MK-801 itself can damage neurons. Here we show that MK-801 (0.1 to 5.0 mg/kg) and ketamine (40 to 100 mg/kg) typically induce heat shock protein HSP72 mainly in layer 3 neurons of the posterior cingulate and retrosplenial cortex of the rat. These HSP72-immunoreactive neurons contain abnormal cytoplasmic vacuoles visualized by electron microscopy. The HSP72 immunoreactivity is maximal at 24 hours with 1.0-mg/kg doses of MK-801 and disappears by 2 weeks. Based on these data, we propose: (1) MK-801 and ketamine injure selected neurons, which express HSP72 in response to that injury. (2) Since HSP72 is induced for 1 to 2 weeks, the prolonged psychological side effects of MK-801, ketamine, phencyclidine, and related drugs could be related to this injury. (3) The neuroprotective effect of MK-801 is probably not related to HSP72 induction. (4) HSP72 immunocytochemistry is useful for studying nonlethal neuronal injury from a wide variety of brain insults.
Epidermal growth factor and transforming growth factor alpha induce c-fos gene expression in retinal Muller cells in vivo.
Epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) are peptides that act at a common receptor and are mitogenic for immature astrocytes and trophic for developing brain neurons in vitro. However, a role for these growth factors in the mature nervous system has not been established. To investigate the actions of EGF and TGF alpha in the adult central nervous system (CNS) in vivo, the growth factors were injected into the vitreous cavity of adult male rabbits. After varying intervals, the retinas were examined for c-fos mRNA by Northern blot hybridization or Fos (and Fos-related antigen) protein by immunocytochemistry. EGF induction of c-fos mRNA occurs within 30 min and persists more than 4 hr. Fos nuclear immunostaining is induced selectively in nuclei of Muller cells by both EGF and TGF alpha. Fos-like immunoreactivity appears within 1 hr and persists more than 9 hr after EGF injection. These observations demonstrate that mature retinal Muller cells respond to exogenously applied EGF and TGF alpha in vivo, although the effect of the growth factors is not necessarily direct. The expression of c-fos and other immediate early genes provides a short-term marker that can be used to investigate the role of growth factors in normal retinal physiology and responses to injury.
N-methyl-D-aspartate receptor activation in the neostriatum increases c-fos and fos-related antigens selectively in medium-sized neurons.
In the neostriatum a selective loss of neurons occurs following exposure to N-methyl-D-aspartate receptor agonists. One hypothesis emerging from this observation is that an excitotoxic process via N-methyl-D-aspartate receptors may contribute to the pathogenesis of Huntington's disease, which is characterized by the loss of medium-sized neurons. However, whether there is a selective distribution of N-methyl-D-aspartate receptors in specific populations of neostriatal neurons is unknown. In this study the expression of c-fos mRNA and protein was used to examine the response of neostriatal cells to N-methyl-D-aspartate receptor stimulation in the rat. After intrastriatal injection of the N-methyl-D-aspartate receptor agonist, quinolinic acid, an increase in c-fos mRNA concentrations was detected using in situ hybridization and Northern blot analysis. Western blot analysis showed that not only the c-Fos mRNA protein product but also other Fos-related antigens capable of binding to DNA were increased in response to N-methyl-D-aspartate receptor activation. The selectivity of the neuronal response to N-methyl-D-aspartate receptor activation was examined immunohistochemically at the light and ultrastructural levels. Our results indicate that N-methyl-D-aspartate receptor activation by quinolinic acid stimulates medium spiny neurons to increase c-Fos expression; to a lesser extent, medium aspiny interneurons and glial cells also respond. In contrast, negligible change in c-Fos expression is observed in large neurons. These results are consistent with other evidence that medium-sized spiny neurons are preferentially vulnerable to the toxic effects of excitatory amino acids acting at N-methyl-D-aspartate receptors. An additional implication of these findings is that activation of the N-methyl-D-aspartate receptor in medium spiny neurons leads to increased expression of candidate AP-1 transcription factors, thereby coupling the N-methyl-D-aspartate receptor and regulation of gene expression in signal transduction processes of the neostriatal medium spiny neuron.
Induction of heat shock protein 72-like immunoreactivity in the hippocampal formation following transient global ischemia.
Global ischemia was produced in adult rats by combining bilateral carotid artery occlusions with systemic hypotension for 5 or 10 minutes. Induction of the 72 kD heat shock protein (HSP72) in the hippocampus was examined immunocytochemically 18-24 hours later. Several patterns of HSP72-like immunoreactivity (HSP72LI) were observed. Five minutes of ischemia induced HSP72 in isolated columns of CA1a pyramidal neurons, or throughout CA1 pyramidal neurons and dentate hilar neurons. Ten minutes of ischemia induced marked HSP72LI in CA3 pyramidal neurons, moderate HSP72LI in dentate granule cells, and minimal HSP72LI in CA1 pyramidal, dentate hilar neurons, and hippocampal glia. Two hippocampi subjected to 10 minutes of ischemia exhibited marked HSP72LI in capillary endothelial cells but no neuronal or glial HSP72LI. It is proposed that (a) the induction of HSP72 in hippocampal sectors correlates with their vulnerability to global ischemia (CA1 greater than hilus greater than CA3 greater than dentate gyrus); (b) the induction of HSP72 in hippocampal cells correlates with their vulnerability to global ischemia in that mild ischemia induced HSP72 only in neurons, moderate ischemia in neurons and glia, and severe ischemia only in capillary endothelial cells; (c) the failure to induce HSP72 in hippocampal neurons in 2 cases of 10 min ischemia may be related to severe injury causing disruption of protein synthesis in these cells.
Heat shock protein hsp72 induction in cortical and striatal astrocytes and neurons following infarction.
Transient global and transient focal ischemia induced the 72 kDa heat shock protein (hsp72) in neurons in cortex, striatum, and other regions known to be injured during transient ischemia. A novel finding was the induction of hsp72 in islands (cylinders in three dimensions) of cells composed of astrocytes around the perimeter and neurons in the interior. Since histology showed pale staining in these regions, it is proposed that these islands represent areas of focal infarction in the distribution of small cortical and lenticulostriate arteries. Although the factors responsible for hsp72 induction during ischemia and infarction are unknown, these results suggest differences in mechanisms of hsp72 induction in astrocytes compared to neurons.
Motor deficits are produced by removing some cortical transplants grafted into injured sensorimotor cortex of neonatal rats.
Fetal frontal cortex was transplanted into cavities formed in the right motor cortex of neonatal rats. As adults, the animals were trained to press two levers in rapid succession with their left forelimb to receive food rewards. Once they had reached an optimal level of performance, the effect of removing their transplants was assessed. Surgical removal of transplants significantly impaired the performance of 2 of 4 subjects. Placing a cross-strain skin graft to induce the immunological rejection of the transplants produced a behavioral deficit in 1 of 2 subjects with complete transplant removal. Skin grafts produced no behavioral effects in four subjects that had surviving transplants. Since the motor deficits produced by transplant removal resembled those observed following the removal of normal motor cortex, we propose that these three transplants functioned within the host brain. Histology showed that the procedures used to remove cortical grafts did not injure any host brains. Therefore, host brain damage is unlikely to account for the behavioral deterioration that followed transplant removals.
c-fos mRNA, Fos, and Fos-related antigen induction by hypertonic saline and stress.
The induction of c-fos mRNA was assessed using Northern blots and in situ hybridization in adult rats administered hypertonic saline (HS) and isotonic saline (IS). HS induced c-fos mRNA in magnocellular paraventricular nucleus (PVNm), parvocellular paraventricular nucleus (PVNp), supraoptic nucleus (SON), and lamina terminalis (LMT). This occurred within 5 min, peaked at 30-60 min, and disappeared by 180 min. Fos protein, detected using a specific monoclonal antibody, was maximal at 1-2 hr and disappeared 4-8 hr after HS administration. This confirms observations showing that the c-fos gene response is transient even in the presence of a continuing stimulus. In contrast, Fos-like immunoreactivity (FLI), detected using two polyclonal antisera, was observed in PVNm, PVNp, SON, and LMT for 1-24 hr during continuous osmotic stimulation. Moreover, FLI was observable in these structures for 7 d in rats administered HS and allowed to drink water ad libitum beginning 24 hr later. At times greater than 8 hr, FLI presumably represents Fos-related antigens (FRA), proteins immunologically and functionally related to Fos, whose expression is much more prolonged than authentic Fos following the osmotic stimulus. In addition to induction of c-fos expression in regions specifically involved in osmotic regulation, HS injections also induced c-fos in many other forebrain regions. In order to assess the induction of c-fos mRNA due to the "stress" of the injections, rats injected with isotonic saline were compared to uninjected controls. Isotonic saline injections induced c-fos mRNA in the PVNp, anterior hypothalamus, suprachiasmatic nucleus, cingulate gyrus, neocortex, ventral lateral septal nucleus, piriform cortex, hippocampal pyramidal and dentate granule neurons, paraventricular and intralaminar thalamic nuclei, bed nuclei of stria terminalis, cortical and medial amygdaloid nuclei, and other structures. In accord with other work, we interpret this pattern of c-fos expression to result from the stress of handling and injections. Since Fos and FRA probably bind to the promoters of target genes and regulate their expression, they likely mediate biochemical changes in the cells activated by the osmotic and stressful stimuli. Whereas the Fos signal is transient, FRA may act on target genes for the duration of the stimulus or longer.
The pattern of 72-kDa heat shock protein-like immunoreactivity in the rat brain following flurothyl-induced status epilepticus.
The inducible 72-kDa heat shock protein (HSP72) is a highly conserved stress protein that is expressed in CNS cells and may play a role in protection from neural injury. We used a monoclonal antibody to HSP72 and immunocytochemistry to localize HSP72 in the rat brain 24 h following either 30 or 60 min of flurothyl-induced status epilepticus. Sprague-Dawley rats were anesthetized with halothane, paralyzed, and ventilated, and remained normotensive and well oxygenated for the duration of the seizures. Seizure activity was quantified via analysis of the scalp EEG pattern. HSP72-like immunoreactivity (HSP72-LI) was induced in specific brain regions in a graded fashion that correlated, in part, with the duration and degree of seizure activity. Milder seizures produced HSP72-LI limited to layers 2 and 3 of frontoparietal cortex, dentate hilus cells, and CA3 pyramidal neurons. More extensive seizures led to HSP72-LI in layers 2, 3 and 5 of frontoparietal and visual cortex, dentate hilus cells, CA1 and CA3 pyramidal neurons, and certain thalamic and amygdaloid nuclei. These are similar to many, but not all, of the brain regions known to be injured with this model. No HSP72-LI was observed in sham-treated controls or flurothyl-treated animals whose seizures were controlled with pentobarbital. HSP72-LI thus localizes to certain regions of seizure-induced injury, and may provide a sensitive method of detecting neuronal 'stress' or injury relatively soon after status epilepticus. Whether or not HSP72 synthesis plays a protective role in the pathogenesis of seizures, or is only a marker for cell injury, remains to be determined.
Marked neurotrophic effects of diffusible substances released from non-target cerebellar cells on thalamic neurons in culture.
A primary culture of thalamic cells from 6-day-old postnatal rats was co-cultured for 6 days with neocortical or cerebellar cells (neurons and astrocytes) from the same litter using a Transwell mesh system. The survival of thalamic neurons grown on the lower well, which were affected by substances released from cells grown on the upper wells, was remarkably promoted by both neocortical co-cultures (target for thalamic projection neurons) and cerebellar co-cultures (non-target). When the cells were seeded on mesh at lower density, the neurotrophic effects of neocortical co-cultures on thalamic neurons (204% of control) were significantly greater than those of cerebellar co-cultures (138%). When the cells were seeded on mesh at higher density, the effects of cerebellar co-cultures increased dramatically (517% of control), while the neurotrophic effects of neocortical co-cultures did not change. Morphologically, the survival of multipolar-shaped thalamic neurons was remarkably improved, as compared to the survival of monopolar, bipolar, and tripolar-shaped thalamic neurons. Basic fibroblast growth factor slightly promoted thalamic neuronal survival (136%), whereas nerve growth factor had no effect. These results suggest that neocortical and cerebellar cells release diffusible factor(s) that promote the survival of specific subpopulation of thalamic neurons, and that at least one of the non-target cerebellar cell-derived factor(s) might be more potent than those released from target neocortical cells.