Immunohistochemical Localization of Lipocortin 1 in Rat Brain Is Sensitive to pH, Freezing, and Dehydration

Lipocortin 1 (LC1, annexin 1) has received considerable attention as a substrate for protein kinases, as a Ca++- and phosphatidylserine-binding protein, and as a mediator of glucocorticoid anti-inflammatory effects. However, there has been confusion over localization of LC1 immunoreactivity (LC1-ir), which reportedly localizes to neurons and/or to astrocytes or microglia in rat brain. To test whether these contradictory data arise from unusual properties of the antigen, we developed a novel brain slice model to determine fixation and staining variables. The specificity of anti-LC1 sera was ensured by pre-absorption and affinity purification with immobilized recombinant LC1. Specific LC1-ir was detected in ramified microglia of brains perfused with acidified aldehydes and embedded in paraffin. However, commonly used immunohistochemical procedures have unexpected profound effects. LC1-ir was eliminated by fixation with neutral/alkaline aldehydes, by freezing before strong acid-aldehyde fixation, or by staining without partial de/rehydration before the primary serum. The sensitivity of LC1 epitopes to proton and water activities may reflect molecular properties important to LC1's roles in vivo. True LC1-ir was not detected in normal neurons or astrocytes.

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Intracellular pH in rat brain in vivo and in brain slices

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y92-272 ◽

1992 ◽

Vol 70 (S1) ◽

pp. S269-S277 ◽

Cited By ~ 31

Author(s):

Joseph C. LaManna ◽

J. Keven Griffith ◽

Boris R. Cordisco ◽

Chii-Wann Lin ◽

W. David Lust

Keyword(s):

Cardiac Arrest ◽

Rat Brain ◽

Intracellular Ph ◽

Brain Slice ◽

Spatial Information ◽

Brain Slices ◽

Neutral Red ◽

Hydrogen Ion ◽

Sodium Hydrogen

Intracellular pH can be measured quantitatively in rat brain in vivo and in vitro using spectrophotometric detection of the vital dye neutral red. This method preserves spatial information and is compatible with microhistochemistry. The intracellular pH indicated by this method is in close agreement with that indicated by 31P-NMR spectroscopy. During ischemia, intracellular acidification is correlated with tissue lactate accumulation. The spatial distribution of pH values becomes more heterogeneous as the tissue becomes more acidic. Resuscitation from total cerebral ischemia produced by cardiac arrest results in rapid intracellular realkalinization. This realkalinization is at least partially inhibited by amiloride pretreatment. Some neuronal populations, especially in the hippocampal CA1 and CA4 regions, may become more acidic during ischemia and realkalinize more slowly after reperfusion than other tissue regions. The intracellular pH of hippocampal brain slice preparations is more alkaline than expected from in vivo studies. The intracellular pH of the brain slice can be acidified to near neutrality by specific inhibitors of the sodium/hydrogen ion exchanger.Key words: hippocampal brain slice, intracellular pH, neutral red, cardiac arrest and resuscitation, sodium/hydrogen ion exchanger.

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Acute hypoxia induces elevation of ornithine decarboxylase activity in neonatal rat brain slices

Reproduction Fertility and Development ◽

10.1071/rd9950385 ◽

1995 ◽

Vol 7 (3) ◽

pp. 385 ◽

Cited By ~ 2

Author(s):

LD Longo ◽

S Packianathan

Keyword(s):

Rat Brain ◽

Ornithine Decarboxylase ◽

Bovine Serum ◽

Brain Slice ◽

Brain Slices ◽

Neonatal Rat ◽

Newborn Rat ◽

Rat Brain Slice

Recent studies in vivo have demonstrated that ornithine decarboxylase (ODC) activity in the fetal rat brain is elevated 4-5-fold by acute maternal hypoxia. This hypoxic-associated increase is seen in the rat brain in both the newborn and the adult. Because of the intimate involvement of ODC in transcription and translation, as well as in growth and development, it is imperative that the manner in which hypoxia affects the regulation of this enzyme be better understood. In order to achieve this, a brain preparation in vitro was required to eliminate the confounding effects of the dam on the fetal and newborn brain ODC activity in vivo. Therefore, brain slices from 3-4-day-old (P-3) newborn rats were utilized to test the hypothesis that ODC activity increases in response to hypoxia in vitro. Cerebral slices from the P-3 rat pups were allowed to equilibrate and recover in artificial cerebrospinal fluid (ACSF) continuously bubbled with a mixture of 95% O2 and 5% CO2 for 1 h before beginning hypoxic exposures. Higher basal ODC activities were obtained by treating the slices with 0.03% fetal bovine serum (FBS) and 0.003% bovine serum albumin (BSA), rather than with ACSF alone. Hypoxia was induced in the slices by replacing the gas with 40%, 21%, 10%, or 5% O2, all with 5% CO2 and balance N2. With FBS and BSA treatment, ODC activity was maintained at about 0.15-0.11 nM CO2 mg-1 protein h-1 throughout the experiment, which was 2-3-fold higher than that without FBS and BSA. ODC activity increased significantly and peaked between 1 h and 2 h after initiation of hypoxia. For instance, with 21% O2, ODC activity increased approximately 1.5-fold at 1 h and approximately 2-fold at 2 h. These studies demonstrate that: (1) the hypoxic-induced increases observed in vivo in the fetal and newborn rat brain ODC activity can be approximated in a newborn rat brain slice preparation in vitro; (2) newborn rat brain slice preparations may provide an alternative to methods in vivo or cell culture methods for studying the regulation of acute hypoxic-induced enzymes; and (3) high, stable baseline ODC activities in brain slices suggest that the cells in the slice are capable of active metabolism if FBS and BSA are available to mimic conditions in vivo.

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Spindle-Like Thalamocortical Synchronization in a Rat Brain Slice Preparation

Journal of Neurophysiology ◽

10.1152/jn.2000.84.2.1093 ◽

2000 ◽

Vol 84 (2) ◽

pp. 1093-1097 ◽

Cited By ~ 27

Author(s):

Virginia Tancredi ◽

Giuseppe Biagini ◽

Margherita D'Antuono ◽

Jacques Louvel ◽

René Pumain ◽

...

Keyword(s):

Rat Brain ◽

Kynurenic Acid ◽

Brain Slice ◽

Excitatory Amino Acid ◽

Brain Slices ◽

Reticular Nucleus ◽

Antidromic Responses ◽

Rat Brain Slice

We obtained rat brain slices (550–650 μm) that contained part of the frontoparietal cortex along with a portion of the thalamic ventrobasal complex (VB) and of the reticular nucleus (RTN). Maintained reciprocal thalamocortical connectivity was demonstrated by VB stimulation, which elicited orthodromic and antidromic responses in the cortex, along with re-entry of thalamocortical firing originating in VB neurons excited by cortical output activity. In addition, orthodromic responses were recorded in VB and RTN following stimuli delivered in the cortex. Spontaneous and stimulus-induced coherent rhythmic oscillations (duration = 0.4–3.5 s; frequency = 9–16 Hz) occurred in cortex, VB, and RTN during application of medium containing low concentrations of the K+ channel blocker 4-aminopyridine (0.5–1 μM). This activity, which resembled electroencephalograph (EEG) spindles recorded in vivo, disappeared in both cortex and thalamus during application of the excitatory amino acid receptor antagonist kynurenic acid in VB ( n = 6). By contrast, cortical application of kynurenic acid ( n = 4) abolished spindle-like oscillations at this site, but not those recorded in VB, where their frequency was higher than under control conditions. Our findings demonstrate the preservation of reciprocally interconnected cortical and thalamic neuron networks that generate thalamocortical spindle-like oscillations in an in vitro rat brain slice. As shown in intact animals, these oscillations originate in the thalamus where they are presumably caused by interactions between RTN and VB neurons. We propose that this preparation may help to analyze thalamocortical synchronization and to understand the physiopathogenesis of absence attacks.

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Lipocortin-1 is an endogenous inhibitor of ischemic damage in the rat brain.

Journal of Experimental Medicine ◽

10.1084/jem.174.2.305 ◽

1991 ◽

Vol 174 (2) ◽

pp. 305-310 ◽

Cited By ~ 114

Author(s):

J K Relton ◽

P J Strijbos ◽

C T O'Shaughnessy ◽

F Carey ◽

R A Forder ◽

...

Keyword(s):

Cerebral Ischemia ◽

Rat Brain ◽

Therapeutic Potential ◽

Artery Occlusion ◽

Ischemic Damage ◽

Endogenous Inhibitor ◽

Annexin 1 ◽

Marked Inhibition ◽

Cerebral Artery Occlusion

Lipocortin-1 (annexin-1) is an endogenous peptide with antiinflammatory properties. We have previously demonstrated lipocortin immunoreactivity in certain glial cells and neurons in the rat brain (Strijbos, P.J.L.M., F.J.H. Tilders, F. Carey, R. Forder, and N.J. Rothwell. 1990. Brain Res. In press.), and have shown that an NH2-terminal fragment (1-188) of lipocortin-1 inhibits the central and peripheral actions of cytokines on fever and thermogenesis in the rat in vivo (Carey, F., R. Forder, M.D. Edge, A.R. Greene, M.A. Horan, P.J.L.M. Strijbos, and N.J. Rothwell. 1990. Am. J. Physiol. 259:R266; and Strijbos, P.J.L.M., J.L. Browning, M. Ward, R. Forder, F. Carey, M.A. Horan, and N.J. Rothwell. 1991. Br. J. Pharmacol. In press.). We now report that intracerebroventricular administration of lipocortin-1 fragment causes marked inhibition of infarct size (60%) and cerebral edema (46%) measured 2 h after cerebral ischemia (middle cerebral artery occlusion) in the rat in vivo. The lipocortin-1 fragment was effective when administered 10 min after induction of ischemia. Ischemia caused increased expression of lipocortin-1 around the area of infarction as demonstrated by immunocytochemistry. Intracerebroventricular injection of neutralizing antilipocortin-1 fragment antiserum increased the size of infarct (53%) and the development of edema (29%). These findings indicate that lipocortin-1 is an endogenous inhibitor of cerebral ischemia with considerable therapeutic potential.

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Characterization of phospholipid methylation in rat brain myelin

Biochemical Journal ◽

10.1042/bj3070239 ◽

1995 ◽

Vol 307 (1) ◽

pp. 239-244 ◽

Cited By ~ 9

Author(s):

V Tsvetnitsky ◽

L Auchi ◽

A Nicolaou ◽

W A Gibbons

Keyword(s):

Rat Brain ◽

Affinity Purification ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Methyl Transferase ◽

Sds Page ◽

Triton X ◽

Predominant Product

Highly purified rat brain myelin was solubilized in Triton X-100 and myelin phospholipid N-methyltransferase was characterized. The enzyme activities were separated by isoelectric focusing and ion-exchange chromatography. The phospholipid methyl-transferase has shown at least four peaks of activity with pIapp. values of 4.5, 5.2, 6.2 and 8.4. After affinity purification each of these activities revealed a close set of bands of approx. 65 kDa on SDS/PAGE. These data together with those from preparative SDS/PAGE separations suggested that rat brain myelin contains three acidic and at least one basic phospholipid-methylating isoenzymes and that the major isoenzyme in each case is approx. 65 kDa in size. While the predominant product of the reaction catalysed by all detected isoforms was monomethylated phosphatidylethanolamine, the least acidic isoform (pIapp. 6.2) also formed about 20% phosphatidylcholine, suggesting that these isoenzymes may play different roles in vivo.

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