Biomedical subjects

J A Rafols

Publications and source records attributed to J A Rafols.

At least 19 recordsLinked to original sources

PubMed2006

Exercise preconditioning upregulates cerebral integrins and enhances cerebrovascular integrity in ischemic rats.

We hypothesized that exercise preconditioning strengthens brain microvascular integrity against ischemia/reperfusion injury through the tumor necrosis factor (TNF)-integrin signaling pathway. Adult male Sprague Dawley rats (n = 24) were studied in: (1) exercise (the animals run on a treadmill 30 min each day) for 3 weeks, (2) non-exercise. Six animals from each group (n = 12) were subjected to stroke, the remaining animals served as controls (n = 6 x 2). Brain infarction and edema were determined by Nissl staining. Cerebral integrin expression was detected by immunochemistry and stereological methods. In addition, we used flow cytometry to address the causal role of TNF-alpha in inducing the expression of integrins in the human umbilical vein endothelial cells under TNF-alpha or vascular endothelial growth factor (VEGF) pretreatment. Exercise reduces brain infarction and brain edema in stroke. Expressions of integrin subunit alpha(1), alpha(6), beta(1), and beta(4) were increased after exercise. Exercise preconditioning reversed stroke-reduced integrin expression. An in vitro study revealed a causal link between the gradual upregulation of TNF-alpha (rather than VEGF) and cellular expression of integrins. These results demonstrated an increase in cerebral expression of integrins and a decrease in brain injury from stroke after exercise preconditioning. The study suggests that upregulation of integrins during exercise enhances neurovascular integrity after stroke. The changes in integrins might be altered by TNF-alpha.

Animals↗

PubMed2006

Immunohistochemical mapping of total and phosphorylated eukaryotic initiation factor 4G in rat hippocampus following global brain ischemia and reperfusion.

Partial proteolysis and phosphorylation of the translation initiation factor eukaryotic initiation factor 4G (eIF4G) occur in reperfused brain, but the contribution of eIF4G alterations to brain injury has not been established. A component of the complex delivering mRNA to the small ribosomal subunit, eIF4G is also found in stress granules. Stress granules sequester inactive 48S preinitiation complexes during stress-induced translation arrest. We performed double-labeling immunofluorescence histochemistry for total or ser 1108 phosphorylated eIF4G and the stress granule component T-cell internal antigen following normothermic, 10 min cardiac arrest-induced global brain ischemia and up to 4 h reperfusion in the rat. In cornu ammonis (Ammon's horn; CA) 1 at 90 min and 4 h reperfusion, eIF4G staining transformed from a homogeneous to an aggregated distribution. The number of eIF4G-containing stress granules differed between CA1 and CA3 during reperfusion. In hippocampal pyramidal neurons, phosphorylated eIF4G appeared exclusively in stress granules. Supragranular interneurons of the dentate gyrus showed a large increase in cytoplasmic eIF4G(P) following reperfusion. Immunoblot analysis with antisera against different portions of eIF4G showed a large increase in phosphorylated C-terminal eIF4G fragments, suggesting these accumulate in the cytoplasm of dentate gyrus interneurons. Thus, altered eIF4G subcellular compartmentalization may contribute to prolonged translation arrest in CA1 pyramidal neurons. Accumulation of phosphorylated eIF4G fragments may contribute to the vulnerability of dentate interneurons. Ischemia and reperfusion invoke different translational control responses in distinct hippocampal neuron populations, which may contribute to the differential ischemic vulnerabilities of these cells.

Analysis of Variance↗

PubMed2005

Prolonged translation arrest in reperfused hippocampal cornu Ammonis 1 is mediated by stress granules.

Global brain ischemia and reperfusion cause phosphorylation of the alpha subunit of eukaryotic initiation factor 2alpha, a reversible event associated with neuronal translation inhibition. However, the selective vulnerability of cornu Ammonis (CA) 1 pyramidal neurons correlates with irreversible translation inhibition. Phosphorylation of eukaryotic initiation factor 2alpha also leads to the formation of stress granules, cytoplasmic foci containing, in part, components of the 48S pre-initiation complex and the RNA binding protein T cell internal antigen-1 (TIA-1). Stress granules are sites of translationally inactive protein synthesis machinery. Here we evaluated stress granules in rat hippocampal formation neurons after 10 min global brain ischemia and 10 min, 90 min or 4 h of reperfusion by double-labeling immunofluorescence for two stress granule components: small ribosomal subunit protein 6 and TIA-1. Stress granules in CA3, hilus and dentate gyrus, but not CA1, increased at 10 min reperfusion and returned to control levels by 90 min reperfusion. Dynamic changes in the nuclear distribution of TIA-1 occurred in resistant neurons. At 4 h reperfusion, small ribosomal subunit protein 6 was solely localized within stress granules only in CA1 pyramidal neurons. Both TIA-1 and small ribosomal subunit protein 6 levels decreased approximately 50% in hippocampus homogenates. Electron microscopy showed stress granules to be composed of electron dense bodies 100-200 nm in diameter, that were not membrane bound, but were associated with endoplasmic reticulum. Alterations in stress granule behavior in CA1 pyramidal neurons provide a definitive mechanism for the continued inhibition of protein synthesis in reperfused CA1 pyramidal neurons following dephosphorylation of eukaryotic initiation factor 2alpha.

Animals↗

PubMed2004

Intracellular coexpression of endothelin-1 and inducible nitric oxide synthase underlies hypoperfusion after traumatic brain injury in the rat.

We used Marmarou's rat model of traumatic brain injury to demonstrate colocalization of mRNAs for endothelin-1 (ET-1, a powerful vasoconstrictor) and inducible nitric oxide synthase (iNOS, generator of NO, a vasodilator) in individual cells that form the brain's microvascular wall. The results were confirmed with double immunocytochemistry. After trauma endothelial, smooth muscle cells and macrophages contributed to the abnormal synthesis of ET-1 and iNOS which may underlie a dysfunctional brain microcirculation. This is the first in vivo single cell demonstration of ET-1 and iNOS colocalization, suggesting reciprocal regulation of each other's expression both at the transcriptional and translational levels. The results further indicate that interaction between ET-1 and iNOS occurs at the cytosol and possibly the nuclear membranes, implicating mediation via endothelin receptors.

Animals↗

PubMed2004

Motor balance and coordination training enhances functional outcome in rat with transient middle cerebral artery occlusion.

The goal of this study was to determine if relatively complex motor training on Rota-rod involving balance and coordination plays an essential role in improving motor function in ischemic rats, as compared with simple locomotor exercise on treadmill. Adult male Sprague-Dawley rats with (n=40) or without (n=40) ischemia were trained under each of three conditions: (1) motor balance and coordination training on Rota-rod; (2) simple exercise on treadmill; and (3) non-trained controls. Motor function was evaluated by a series of tests (foot fault placing, parallel bar crossing, rope and ladder climbing) before and at 14 or 28 days after training procedures in both ischemic and normal animals. Infarct volume in ischemic animals was determined with Nissl staining. Compared with both treadmill exercised and non-trained animals, Rota-rod-trained animals with or without ischemia significantly (P<0.01) improved motor performance of all tasks except for foot fault placing after 14 days of training, with normal rats having better performance. Animals trained for up to 28 days on the treadmill did not show significantly improved function. With regard to foot fault placing task, performance on foot placing was improved in ischemic rats across the three measurements at 0, 14 and 28 days regardless of training condition, while the normal group reached their best performance at the beginning of measurement. No significant differences in infarct volume were found in rats trained either with Rota-rod (47+/-4%; mean+/-S.E.), treadmill (45+/-5%) or non-exercised control (45+/-3%). In addition, no obvious difference could be detected in the location of the damage which included the dorso-lateral portion of the neostriatum and the frontoparietal cortex, the main regions supplied by the middle cerebral artery. The data suggest that complex motor training rather than simple exercise effectively improves functional outcome.

Animals↗

PubMed2004

Exercise pre-conditioning reduces brain damage in ischemic rats that may be associated with regional angiogenesis and cellular overexpression of neurotrophin.

There is increasing evidence that physical activity is associated with a decreased stroke risk. The purpose of this study was to determine if exercise could also reduce brain damage in rats subjected to transient middle cerebral artery (MCA) occlusion, and if the reduced brain injury is associated with angiogenesis as well as cellular expression of the nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in regions supplied by the MCA. Adult male Sprague Dawley rats (n=36) exercised 30 min each day for 3 weeks on a treadmill on which repetitive locomotor movement was required. Then, stroke was induced by a 2-h MCA occlusion using an intraluminal filament, followed by 48 h of reperfusion. In addition to the two exercised groups of animals with or without MCA occlusion, there were two other groups of animals, with or without MCA occlusion, housed for the same duration and used as non-exercised controls. Brain damage in ischemic rats was evaluated by neurologic deficits and infarct volume. Exercise preconditioned and non-exercised brains were processed for immunocytochemistry to quantify the number of microvessels or NGF- and BDNF-labeled cells. Pre-ischemic motor activity significantly (P<0.01) reduced neurologic deficits and infarct volume in the frontoparietal cortex and dorsolateral striatum. Cellular expressions of NGF and BDNF were significantly (P<0.01) increased in cortex (neuron) and striatum (glia) of rats under the exercise condition. Significant (P<0.01) increases in microvessel density were found in striatum. Physical activity reduced stroke damage. The reduced brain damage may be attributable to angiogenesis and neurotrophin overexpression in brain regions supplied by the MCA following exercise.

Animals↗

PubMed2004

Attenuation of iNOS mRNA exacerbates hypoperfusion and upregulates endothelin-1 expression in hippocampus and cortex after brain trauma.

Nitric oxide (NO, a vasodilator) and endothelin-1 (ET-1, a powerful vasoconstrictor) participate in the regulation of brain's microcirculation influencing each other's expression and synthesis. Following injury to the brain, NO is derived largely from the inducible form of nitric oxide synthase (iNOS). We used Marmarou's model of traumatic brain injury (TBI) to study the cerebral blood flow and expression (mRNA) of ET-1 in rats that were pretreated with antisense iNOS oligodeoxynucleotides (ODNs). Intracerebroventricular application of iNOS ODNs resulted in reduced synthesis of iNOS as detected by Western blot analysis. The cerebral blood flow (measured by laser Doppler flowmetry), generally decreased after TBI, was further markedly reduced in the treated animals and remained at low levels up to 48 h post-TBI. The expression of ET-1 (detected by in situ hybridization in cortex and hippocampus) was increased 2-3-fold following TBI alone and this increase reached 5-6-fold in animals pretreated with antisense iNOS ODNs. The results indicate that most likely, NO, generated primarily by iNOS, suppresses ET-1 production and that a decrease of NO results in upregulation of ET-1 via transcriptional and translational mechanisms. Increased availability of ET-1 at the vascular bed and the neuropil may contribute to the altered microvascular reactivity and reduced perfusion of the brain following TBI.

Animals↗

PubMed2002

Sources of endothelin-1 in hippocampus and cortex following traumatic brain injury.

Endothelin 1 (ET-1) exerts normally a powerful vasoconstrictor role in the control of the brain microcirculation. In altered states, such as following traumatic brain injury (TBI), it may contribute to the development of ischemia and/or secondary cell injury. Because little is known of ET-1's cellular compartmentalization and its association to vulnerable neurons after TBI, we assessed its expression (both mRNA and protein) in cerebral cortex and hippocampus using correlative in situ hybridization and immunocytochemical techniques.Sprague-Dawley male rats were killed at 4, 24 or 48 h after TBI (450 g from 2 m, Marmarou's model). Semiquantitative analysis of our in situ hybridization results indicated a 2.5- and a 2.0-fold increase in ET-1 mRNA content in the hippocampus and cortex respectively which persisted up to 48 h post TBI. At 4 and 24 h after TBI enzyme-linked immunosorbent assay showed a tendency for increased ET-1 synthesis. In animals subjected to TBI, qualitative immunocytochemical analysis revealed a shift in ET-1 expression from astrocytes (in control animals) to endothelial cells, macrophages and neurons. Astrocytes and macrophages were identified unequivocally by using double immunofluorescence revealing ET-1 and glial fibrillary acidic protein or ED-1, respectively, the markers being specific for these cellular types. While this redistribution was most prominent at 4 and 24 h post TBI, at 48 h the endothelial cells remained strongly ET-1 immunopositive. The results suggest that cellular types which in the intact animal synthesize little or no ET-1 provide novel sources of the peptide after TBI. These sources may contribute to the sustained cerebrovascular hypoperfusion observed post TBI.

Animals↗

PubMed2001

Upregulation of iNOS expression and phosphorylation of eIF-2alpha are paralleled by suppression of protein synthesis in rat hypothalamus in a closed head trauma model.

When the inducible form of nitric oxide synthase (iNOS) is expressed after challenge to the nervous system, it results in abnormally high concentrations of nitric oxide (NO). Under such conditions, NO could phosphorylate the eukaryotic translation initiation factor (eIF)-2alpha, thus suppressing protein synthesis in neurons that play a role in endocrine and autonomic functions. Using the Marmarou model of traumatic brain injury (TBI), we observed a rapid increase (at 4 h after TBI) of iNOS mRNA in magno- and parvocellular supraoptic and paraventricular neurons, declining gradually by approximately 30% at 24 h and by approximately 80% at 48 h. Western analysis indicated a trend towards increased iNOS protein synthesis at 4 h, which peaked at 8 h, and tended to decrease at the later time points. At the same time points, we detected immunocytochemically the phosphorylated form of eIF-2alpha (eIF-2alpha[P]) as cytoplasmic and more often as nuclear labeling. The incidence of double-labeled [iNOS and eIF-2alpha(P)] neuronal profiles, particularly at 24 h and 48 h after TBI, was high. De novo protein synthesis assessed quantitatively after infusion of 35S methionine/cysteine was reduced by approximately 20% at 4 h, remained depressed at 24 h, and did not return to control levels up to 48 h following the trauma. The results suggest that iNOS may trigger phosphorylation of eIF-2alpha, which in turn interferes with protein synthesis at the translational (ribosomal complex) and transcriptional (chromatin) levels. The depression in protein synthesis may include downregulation of iNOS itself, which could be an autoregulatory inhibitory feedback mechanism for NO synthesis. Excessive amounts of NO may also participate in dysfunction of hypothalamic circuits that underlie endocrine and autonomic alterations following TBI.

Animals↗

PubMed2001

GnRH and gonadotropin release is decreased in chronic nitric oxide deficiency.

Nitric oxide synthetase (NOS), the conversion enzyme for nitric oxide (NO) is localized in the anterior pituitary of female rats, particularly in gonadotrophs and folliculo-stellate cells, suggesting that NO regulates the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from the anterior pituitary. The focus of this study was to determine the effect of chronic NO deficiency on the subsequent pituitary release of LH and FSH in vitro and the hypothalamic immunoexpression of GnRH in vivo. NO deficiency was induced by adding the NOS inhibitor, N-nitro-L-arginine (L-NNA, 0.6 g/L) to the drinking water of female Wistar rats. After 8 weeks, the animals were euthanized, the pituitaries were removed, and they were incubated in vitro. Pituitaries were perfused for 4 hr in the presence of pulsatile gonadotropin release hormone (GnRH, 500 ng/pulse) every 30 min. S-Nitroso-L-acetyl penicillamine (SNAP, an NO donor, 0.1 mM) or L-nitro-argine methyl ester (L-NAME, a NOS inhibitor, 0.1 mM) was added to the media and perfusate samples were collected at 10-min intervals. LH and FSH levels in the perfusate were measured by double antibody radioimmunoassays. Pituitaries from the NO-deficient rats had a significantly smaller GnRH-stimulated release of LH and FSH compared with proestrous control rats. The addition of S-NAP to the perfusate resulted in decreased LH and FSH secretion in the control group, but increased LH secretion in the NO-deficient group. The addition of L-NAME to the perfusate suppressed LH secretion from control pituitaries, but not in pituitaries from NO-deficient animals. Immunohistochemistry of brain slices demonstrated that NO-deficient rats had a large qualitative decrease of GnRH in the median eminence compared with their controls. This decrease was particularly evident in the external capillary plexus of the median eminence. We concluded that chronic NO deficiency is associated with a decreased GnRH in neurosecretory terminals in the external capillary layer of the median eminence, accompanied by a decrease in LH and FSH release from the pituitaries.

Animals↗

PubMed2000

Brain ischemia and reperfusion: molecular mechanisms of neuronal injury.

Brain ischemia and reperfusion engage multiple independently-fatal terminal pathways involving loss of membrane integrity in partitioning ions, progressive proteolysis, and inability to check these processes because of loss of general translation competence and reduced survival signal-transduction. Ischemia results in rapid loss of high-energy phosphate compounds and generalized depolarization, which induces release of glutamate and, in selectively vulnerable neurons (SVNs), opening of both voltage-dependent and glutamate-regulated calcium channels. This allows a large increase in cytosolic Ca(2+) associated with activation of mu-calpain, calcineurin, and phospholipases with consequent proteolysis of calpain substrates (including spectrin and eIF4G), activation of NOS and potentially of Bad, and accumulation of free arachidonic acid, which can induce depletion of Ca(2+) from the ER lumen. A kinase that shuts off translation initiation by phosphorylating the alpha-subunit of eukaryotic initiation factor-2 (eIF2alpha) is activated either by adenosine degradation products or depletion of ER lumenal Ca(2+). Early during reperfusion, oxidative metabolism of arachidonate causes a burst of excess oxygen radicals, iron is released from storage proteins by superoxide-mediated reduction, and NO is generated. These events result in peroxynitrite generation, inappropriate protein nitrosylation, and lipid peroxidation, which ultrastructurally appears to principally damage the plasmalemma of SVNs. The initial recovery of ATP supports very rapid eIF2alpha phosphorylation that in SVNs is prolonged and associated with a major reduction in protein synthesis. High catecholamine levels induced by the ischemic episode itself and/or drug administration down-regulate insulin secretion and induce inhibition of growth-factor receptor tyrosine kinase activity, effects associated with down-regulation of survival signal-transduction through the Ras pathway. Caspase activation occurs during the early hours of reperfusion following mitochondrial release of caspase 9 and cytochrome c. The SVNs find themselves with substantial membrane damage, calpain-mediated proteolytic degradation of eIF4G and cytoskeletal proteins, altered translation initiation mechanisms that substantially reduce total protein synthesis and impose major alterations in message selection, down-regulated survival signal-transduction, and caspase activation. This picture argues powerfully that, for therapy of brain ischemia and reperfusion, the concept of single drug intervention (which has characterized the approaches of basic research, the pharmaceutical industry, and clinical trials) cannot be effective. Although rigorous study of multi-drug protocols is very demanding, effective therapy is likely to require (1) peptide growth factors for early activation of survival-signaling pathways and recovery of translation competence, (2) inhibition of lipid peroxidation, (3) inhibition of calpain, and (4) caspase inhibition. Examination of such protocols will require not only characterization of functional and histopathologic outcome, but also study of biochemical markers of the injury processes to establish the role of each drug.

Adenosine Triphosphate↗

PubMed2000

Pericyte migration from the vascular wall in response to traumatic brain injury.

Any perturbation of the blood brain barrier, whether from changes in cell physiology or from direct injury, may result in microvascular dysfunction and disease. We examined, at the ultrastructural level, microvascular pericyte responses in a well-defined model of traumatic brain injury in the rat. In areas close to the site of impact cortical pericytes underwent a number of changes within the first hour. Approximately 40% of pericytes migrated from their microvascular location. Migration occurred concomitant with a thinning of the abluminal surface of the basal lamina and an accumulation of the receptor for the urokinase plasminogen activator on the leading surface of the migrating cell. Migrated pericytes appeared viable and remained in a perivascular location in the adjacent neuropil. Nonmigrating pericytes in the same section displayed cytoplasmic alterations and nuclear chromatin changes consistent with a rapid degenerative process.

Animals↗

PubMed2000

Expression of the inducible nitric oxide synthase in distinct cellular types after traumatic brain injury: an in situ hybridization and immunocytochemical study.

The Marmarou's acceleration traumatic brain injury (TBI) model, in situ hybridization and immunocytochemistry were utilized to study the temporal expression of the inducible form of nitric oxide synthase (iNOS) mRNA and protein in different cellular compartments of the rat brain. Four hours following TBI, expression of iNOS was observed in the endothelial cells of cerebral blood vessels, macrophages and many cortical and hippocampal neurons. In the cortex labeled neuronal and nonneuronal cells were primarily found in the superficial layers. In the hippocampus the strongest neuronal labeling was observed in the CAI and CA3 (lateral part) regions. By 24 h post TBI endothelial cells no longer expressed iNOS mRNA, and the macrophage and neuronal iNOS expression was reduced by 30-50%. The reduction was assessed by automated quantitation of the silver grains that occupy individual cellular profiles using an image analysis system. Immunocytochemistry revealed de novo iNOS synthesis in non-neuronal cells at the different time points, thus paralleling the changes in iNOS mRNA expression. In contrast, iNOS immunoreactivity in neurons was not observed before 24 h post TBI, suggesting failure of iNOS protein translation at 4 h after trauma. The results demonstrate complex spatial and temporal patterns of iNOS expression in discrete cellular populations, indicating different times of nitric oxide synthesis (and release) following TBI. Uncoupling of iNOS mRNA and protein synthesis in neurons suggests differential synthesis of nitric oxide in these cells as compared to non-neuronal cellular populations after trauma.

Animals↗

PubMed1999

Ultrastructural localization of phosphorylated eIF2alpha [eIF2alpha(P)] in rat dorsal hippocampus during reperfusion.

During post-ischemic brain reperfusion there is a substantial reduction of protein synthesis in selectively vulnerable neurons. Normal protein synthesis requires a functional translation initiation complex, a key element of which is eukaryotic initiation factor 2 (eIF2), which in a complex with GTP introduces the met-tRNA(i). Phosphorylation of Ser(51) on the alpha subunit of eIF2 [eIF2alpha(P)] generates a competitive inhibitor of eIF2B, thereby preventing the replenishment of GTP onto eIF2, thus blocking translation initiation. It has been shown that the conditional expression of an eIF2alpha mutant (Asp substituted for Ser(51)) imitating the negative charge of Ser(51) (P) induces apoptosis. During the first 10 min of post-ischemic reperfusion, there is an approximately 20-fold increase in eIF2alpha(P) seen in the cytoplasm of CA1 hippocampal neurons, and, by 1 h, there is also accumulation of eIF2alpha(P) in the nucleus. We utilized post-embedding electron microscopical immunogold methods to examine the localization of eIF2alpha(P) during reperfusion. Immunogold particles (10 nm) were concentrated chiefly along the rough endoplasmic reticulum and in association with the membranes of the nuclear envelope in CA1 neurons. Aggregations of gold particles in the nucleus were concentrated: (1) within and around the nucleolus, (2) associated to strands of heterochromatin, and (3) along putative nuclear filaments. The presence of eIF2alpha(P) in the nucleolus probably reflects its association with nascent ribosomal subunits. The beta-subunit of eIF2 has a zinc finger and polylysine blocks analogous to those on other proteins that affect transcription. The association of eIF2alpha(P) with chromatin may have important implications for transcription.

Animals↗

PubMed1999

Cell death, calcium mobilization, and immunostaining for phosphorylated eukaryotic initiation factor 2-alpha (eIF2alpha) in neuronally differentiated NB-104 cells: arachidonate and radical-mediated injury mechanisms.

These experiments examine the effects of arachidonate with respect to cell death, radical-mediated injury, Ca2+ mobilization, and formation of ser-51-phosphorylated eukaryotic initiation factor 2alpha [eIF2alpha(P)]. It is known that during brain ischemia the concentration of free arachidonate can reach 180 microM, and during reperfusion oxidative metabolism of arachidonate leads to generation of superoxide that can reduce stored ferric iron and promote lipid peroxidation. During early brain reperfusion, we have shown an approximately 20-fold increase in eIF2alpha(P) which maps to vulnerable neurons that display inhibition of protein synthesis. Here in neuronally differentiated NB-104 cells, equivalent cell death (assessed by LDH release) was induced by 40 microM arachidonate and 20 microM cumene hydroperoxide (CumOOH, a known alkoxyl radical generator). In these injury models (1) radical inhibitors (BHA, BHT, and the lipophilic iron chelator EMHP) block CumOOH-induced cell death but do not block arachidonate-induced death; (2) 40 microM arachidonate (but not up to 40 microM CumOOH) rapidly induces Ca2+ release from intracellular stores; (3) both 40 microM arachidonate and 20 microM CumOOH induce intense immunostaining for eIF2alpha(P); and (4) the elF2alpha(P) immunostaining induced by CumOOH but not that induced by arachidonate is completely blocked by anti-radical intervention with EMHP. Arachidonate-induced formation of eIF2alpha(P) and cell death do not require iron-mediated radical mechanisms and are associated with Ca2+ release from intracellular stores; however, radical-mediated injury also induces both eIF2alpha(P) and cell death without release of intracellular Ca2+. Our data link eIF2alpha(P) formation during brain reperfusion to two established injury mechanisms that may operate concurrently.

Animals↗

PubMed1999

Insulin induces dephosphorylation of eukaryotic initiation factor 2alpha and restores protein synthesis in vulnerable hippocampal neurons after transient brain ischemia.

Brain reperfusion causes prompt, severe, and prolonged protein synthesis suppression and increased phosphorylation of eukaryotic initiation factor 2alpha [eIF2alpha(P)] in hippocampal CA1 and hilar neurons. The authors hypothesized that eIF2alpha(P) dephosphorylation would lead to recovery of protein synthesis. Here the effects of insulin, which activates phosphatases, were examined by immunostaining for eIF2alpha(P) and autoradiography of in vivo 35S amino acid incorporation. Rats resuscitated from a 10-minute cardiac arrest were given 0, 2, 10 or 20 U/kg of intravenous insulin, underwent reperfusion for 90 minutes, and were perfusion fixed. Thirty minutes before perfusion fixation, control and resuscitated animals received 500 microCi/kg of 35S methionine/cysteine. Alternate 30-microm brain sections were autoradiographed or immunostained for eIF2alpha(P). Controls had abundant protein synthesis and no eIF2alpha(P) in hippocampal neurons. Untreated reperfused neurons in the CA1, hilus, and dentate gyrus had intense staining for eIF2alpha(P) and reduced protein synthesis; there was little improvement with treatment with 2 or 10 U/kg of insulin. However, with 20 U/kg of insulin, these neurons recovered protein synthesis and were free of eIF2alpha(P). These results show that the suppression of protein synthesis in the reperfused brain is reversible; they support a causal association between eIF2alpha(P) and inhibition of protein synthesis, and suggest a mechanism for the neuroprotective effects of insulin.

Animals↗

PubMed1998

Organization of the medial pulvinar nucleus in the macaque.

BACKGROUND: The medial pulvinar appears to subserve the integration of associative cortical information and projects to visuomotor-related cortex. In contrast to the other pulvinar subdivisions, the medial pulvinar is a polymodal structure. Therefore, we studied the structural organization of the medial pulvinar to determine how it differs from the surrounding unimodal nuclei. METHODS: Nissl-stained sections were examined to determine the boundaries of, and the distribution of neuronal sizes within, the medial pulvinar. In addition, Golgi-impregnated neurons were examined and drawn for analysis. Only rhesus monkey specimens were used, and the material had been prepared previously for other studies. RESULTS: Projection neurons have round to oval somata and moderate numbers of primary dendrites that extend for short distances before branching into many secondary branches. Two variations of projection neurons (P1 and P2) were distinguished on the basis of the diameters of their dendritic tree. Both varieties have short dendrites that radiate in all directions. They differ in that P2 cells have longer second tier dendrites than P1 cells. Three types of local circuit neurons, tufted, radiating and varicose, were distinguished on the basis of their dendritic morphology. Four types of afferent fibers were identified. Type 1 afferents form cone-shape terminal arbors. Type 2 afferents are similar to those reported for retinal or cortical terminals. Type 3 afferents are of medium thickness and of an unknown origin. Type 4 afferents are thin and have small varicosities consistent with previously described cortical afferents. Afferent fibers are predominantly oriented along the mediolateral axis of the nucleus. We observed putative contacts between some afferents and local circuit neurons and between local circuit neurons and projection neurons. CONCLUSIONS: Medial pulvinar neurons are generally smaller and rounder than those found in the adjacent pulvinar nuclei. These results provide additional evidence for structural distinctions between thalamic nuclei having different functions. However, the observed differences are subtle. In addition, the data in this report provide morphological evidence that cortical signals are likely to be integrated by means of the circuitry located within the nucleus.

Animals↗

PubMed1998

Disruption of MAP-2 immunostaining in rat hippocampus after traumatic brain injury.

The effects of diffuse brain injury on dendritic morphology in rat hippocampus and cortex were examined in this study using the recently described impact acceleration model of traumatic brain injury (Marmarou et al., 1994). Dendritic structure was visualized using immunostaining of microtubule associated protein-2 (MAP-2). Brains were studied 24, 48, and 72 h after brain injury. Results from immunohistochemistry and light microscopy indicated a time-dependent disruption of dendritic cytoarchitecture in the CA1 subregion and in the hilus of the hippocampus but not in the dentate gyrus or CA3 subregion. Similar disruption was observed in the cortical mantle overlying the hippocampus. Although disruption of dendritic structure was observed at 24 h, the most severe damage was at 48 h after injury with evidence of at least partial recovery of MAP-2 immunostaining by 72 h. In the most severe damage, dendrites appeared to be fragmented, scattered, and unaligned, consisting of irregularly spaced and darkly stained swollen segments. A mixed pattern of immunostaining was observed in somata of hilar cells, with some appearing normal while others stained only faintly, appearing to have lost their typical polygonal shape. Semiquantitative rankings confirmed these qualitative findings. Immediate post-injury behavioral evaluations of injury severity were compared to the degree of disruption of MAP-2 immunostaining. The results of this study indicate that diffuse brain injury is associated not only with axonal damage but also with injury to dendrites.

Animals↗