Changes in brain organic osmolytes in experimental cerebral ischemia

doi:10.1016/S0022-510X(98)00062-8

Journal of the Neurological Sciences

Volume 157, Issue 1, 15 April 1998, Pages 25-30

https://doi.org/10.1016/S0022-510X(98)00062-8 Get rights and content

Abstract

The cell volume is regulated not only by inorganic ions, but also by organic osmolytes, such as amino acids, methylamines, and polyhydric alcohols (polyols). Using proton nuclear magnetic resonance spectroscopy (¹H-NMR), we measured the tissue concentrations of amino acids (alanine, aspartate, γ-aminobutyric acid (GABA), glutamate, glutamine, N-acetyl-aspartate (NAA), taurine), methylamines (glycerophosphorylcholine (GPC), creatine+phosphocreatine (total creatine, tCr)), and polyols (myo-inositol) in the rat brain after middle cerebral artery occlusion (incomplete focal ischemia) or after decapitation (complete global ischemia). The total osmolytes expressed as a sum of total amino acids, total methylamines, and total polyols were significantly decreased at 24 h of focal ischemia (58.7% of control value, P=0.0025) whereas they were not changed following decapitation. The water content was increased from control value of 77.9%–84.1% after focal ischemia (P<0.0001) but not after decapitation. These results suggest that the brain organic osmolytes are involved in the process of edema formation following focal cerebral ischemia. Further elucidation of the cellular mechanisms regulating these organic osmolytes in cerebral ischemia may promote greater understanding of the pathophysiology involved in the evolution of brain edema.

Introduction

The role of inorganic ions in the formation of brain edema has been studied extensively 1, 11, 17, 18, 25. Recent studies, however, have demonstrated that the tissue volumes are regulated by a complex mechanism involving not only inorganic ions but also a number of organic solutes (organic osmolytes) such as amino acids (alanine, aspartate, γ-aminobutyric acid, glutamate, glutamine, N-acetyl aspartate, and taurine), methylamines (glycerophosphorylcholine and creatine), and polyhydric alcohols (myo-inositol) 4, 6, 21, 23. When brain swelling developed in an induced systemic hyponatremia, for example, the contents of tissue organic osmolytes were decreased, which potentially ameliorated the swelling of the brain 4, 21. The decrease in brain organic osmolytes may account for approximately 35% of the total decrease of brain solutes [23]. The involvement of organic osmolytes in the process of central pontine myelinolysis has also been suggested by Lien, who explained that this pathologic process is a result of untimely adaptation of local organic osmolytes against the rapid correction of hyponatremia [9].

Despite the detailed information on the changes in inorganic electrolytes during cerebral ischemia 1, 11, 17, 18, 25, little is known about the changes in brain organic osmolytes. We investigated the dynamic changes in organic osmolytes in the rat brains under conditions of focal or global ischemia. Incomplete focal cerebral ischemia was produced by occlusion of the middle cerebral artery (MCA), and complete global ischemia was produced by decapitation. The amounts of organic osmolytes were determined by proton nuclear magnetic resonance (¹H-NMR) spectra, which identify multiple classes of organic compounds in a single sample 3, 6, 12, 14.

Section snippets

Focal ischemia model

Male Sprague-Dawley rats weighing 300–400 g were anesthetized with 300 mg/kg chloral hydrate given intraperitoneally. Rectal temperature was maintained at 37.0±0.5°C with a heating pad. A polyethylene catheter was placed in the left femoral artery for blood pressure measurement and blood sampling. Measurement of arterial blood gas concentration and pH were made at regular intervals. Incomplete focal cerebral ischemia was produced by occlusion of the left MCA using a silicone rubber cylinder

Results

There were no significant differences among the experimental groups with regard to the physiologic variables measured, i.e. rectal temperature, arterial P_aO₂, P_aCO₂, pH, and mean arterial blood pressure.

In the focal ischemia group, the histologic examination of the sampled areas for ¹H-NMR spectroscopy demonstrated tissue swelling and preservation of cellular structures at 2 h of ischemia. At 6 h of ischemia, eosinophilic neurons appeared and the edema became pronounced. The neurons became

Discussion

In this study, ¹H-NMR spectroscopy demonstrated the changes in the major organic solutes in brains subjected to focal ischemia or decapitation. In the focal ischemia model, the brain concentrations of aspartate, glutamate, and GABA decreased and only alanine increased. The sum of these amino acids was decreased significantly. A previous study has demonstrated an increase in the interstitial concentrations of aspartate, GABA, and glutamate following MCA occlusion [10]. The decrease in total

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The proper functioning of any protein depends on its three dimensional conformation which is achieved by the accurate folding mechanism. Keeping away from the exposed stress conditions leads to cooperative unfolding and sometimes partial folding, forming the structures like protofibrils, fibrils, aggregates, oligomers, etc. leading to several neurodegenerative diseases like Parkinson's disease, Alzheimer's, Cystic fibrosis, Huntington, Marfan syndrome, and also cancers in some cases, too. Hydration of proteins is necessary, which may be achieved by the presence of organic solutes called osmolytes within the cell. Osmolytes belong to different classes in different organisms and play their role by preferential exclusion of osmolytes and preferential hydration of water molecules and achieves the osmotic balance in the cell otherwise it may cause problems like cellular infection, cell shrinkage leading to apoptosis and cell swelling which is also the major injury to the cell. Osmolyte interacts with protein, nucleic acids, intrinsically disordered proteins by non-covalent forces. Stabilizing osmolytes increases the Gibbs free energy of the unfolded protein and decreases that of folded protein and vice versa with denaturants (urea and guanidinium hydrochloride). The efficacy of each osmolyte with the protein is determined by the calculation of m value which reflects its efficiency with protein. Hence osmolytes can be therapeutically considered and used in drugs.
Neurochemical changes in unilateral cerebral hemisphere during the subacute stage of focal cerebral ischemia-reperfusion in rats: An ex vivo <sup>1</sup> H magnetic resonance spectroscopy study
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High-resolution nuclear magnetic resonance (NMR) based metabolomic has proved to be very useful for studying ischemic stroke injury (Jiang et al., 2011; Jung et al., 2011). Metabolic disturbance serves as a potential mechanism of ischemia, including elevation of acetate and decreases of Glu and aspartate (Asp) in the ischemic tissue 6 h after occlusion (Håberg et al., 2009; Nonaka et al., 1998). Wang et al. observed 13 metabolites changed significantly in the serum of ischemic stroke injury, malonic acid and glycine (Gly) are the most noticeable variable metabolites (Wang et al., 2014c).
Understanding the subacute may shed light on the mechanism of cerebral ischemia. The present study aimed to explore metabolic features underlying subacute stage of ischemia-reperfusion injury and developing effective treatments. Rats were divided into three groups: the permanent middle cerebral artery occlusion (pMCAO), transient cerebral focal ischemia (tMCAO) and sham group. Evaluation of animal models was performed by the neurological deficit, MR images and pathological morphological abnormality. To elucidate metabolic changes, we conducted a comparative analysis of metabolic composition of unilateral brain tissue using ¹H nuclear magnetic resonance spectroscopy. The successful model was observed low signal on T1WI and high signal on T2WI lesions in the left cerebral. Histopathological results confirmed the formation of apparent lesions in the left striatum, hippocampus CA1 and cortex tissues of subacute cerebral ischemia rats and showed that rats with focal cerebral ischemia-reperfusion could alleviate the extent of pathological damage degree. In pMCAO rats 7 days after surgery, decreased levels of N-acetyl aspartate (NAA), γ-aminobutyric acid (GABA), glutamate (Glu) and succinate (Suc) concomitantly with increased levels of glutamine (Gln), myo-inositol (m-Ins) and lactate (Lac) were observed compared to the control. Whereas, increased level of Lac with decreased levels of NAA, GABA, Glu, Suc, creatine (Cre) were observed in the tMCAO rats. This demonstrated that experimental subacute ischemic stroke in rats caused extensive perturbation in energy metabolism, the tricarboxylic acid cycle and GABA shunt, which provided essential information for understanding the pathogenesis of subacute cerebral ischemia-reperfusion and provided guidance in choosing the suitable therapeutic schedule.
Rapamycin ameliorates brain metabolites alterations after transient focal ischemia in rats
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Rapamycin has been shown to protect against middle cerebral artery occlusion (MCAo) induced ischemic injury. In this study, the neuroprotective effect of rapamycin on the metabolic changes induced by MCAo was evaluated using nuclear magnetic resonance (NMR) spectroscopy of brain tissues. MCAo in rats was induced by insertion of nylon filament. One hour after ischemia, rapamycin (250 µg/kg, i.p.) in dimethyl sulfoxide was administered. Reperfusion was done 2 h after ischemia. Twenty-four hours after ischemia phospholipase A₂ (PLA₂) levels and metabolic changes were assessed. Perchloric acid extraction was performed on the brain of all animals (n=7; sham, vehicle; DMSO and rapamycin 250 µg/kg) and the various brain metabolites were assessed by NMR spectroscopy. In all 44 metabolites were assigned in the proton NMR spectrum of rat brain tissues. In the vehicle group, we observed increased lactate levels and decreased levels of glutamate/glutamine, choline containing compounds, creatine/phosphocreatine (Cr/PCr), taurine, myo-inositol, γ-amino butryic acid (GABA), N-aspartyl aspartate (NAA), purine and pyrimidine metabolites. In rapamycin treated rats, there was increase in the levels of choline containing compounds, NAA, myo-inositol, glutamate/glutamine, GABA, Cr/PCr and taurine as compared to those of vehicle control (P<0.05). Rapamycin treatment reduced PLA₂ levels as compared to vehicle group (P<0.05). Our findings indicated that rapamycin reduced the increased PLA₂ levels and altered brain metabolites after MCAo. These protective effects might be attributed to its effect on cell membrane metabolism; glutamate induced toxicity and calcium homeostasis in stroke.
Increased brain lactate is central to the development of brain edema in rats with chronic liver disease
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In the context of HE, increased glutamine levels are commonly observed even when brain edema is not present, as observed in portacaval-shunted rats [45]. However, in other neurological diseases such as cerebral ischemia, increased brain lactate levels have been associated with the development of brain edema, while brain glutamine levels were decreased [50–52]. Although our study was performed in frontal cortex, it is possible other cerebral regions may be affected dissimilarly.
The pathogenesis of brain edema in patients with chronic liver disease (CLD) and minimal hepatic encephalopathy (HE) remains undefined. This study evaluated the role of brain lactate, glutamine and organic osmolytes, including myo-inositol and taurine, in the development of brain edema in a rat model of cirrhosis.
Six-week bile-duct ligated (BDL) rats were injected with ¹³C-glucose and de novo synthesis of lactate, and glutamine in the brain was quantified using ¹³C nuclear magnetic resonance spectroscopy (NMR). Total brain lactate, glutamine, and osmolytes were measured using ¹H NMR or high performance liquid chromatography. To further define the interplay between lactate, glutamine and brain edema, BDL rats were treated with AST-120 (engineered activated carbon microspheres) and dichloroacetate (DCA: lactate synthesis inhibitor).
Significant increases in de novo synthesis of lactate (1.6-fold, p <0.001) and glutamine (2.2-fold, p <0.01) were demonstrated in the brains of BDL rats vs. SHAM-operated controls. Moreover, a decrease in cerebral myo-inositol (p <0.001), with no change in taurine, was found in the presence of brain edema in BDL rats vs. controls. BDL rats treated with either AST-120 or DCA showed attenuation in brain edema and brain lactate. These two treatments did not lead to similar reductions in brain glutamine.
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Elevation of acetate and decreases of glutamine and aspartate were also observed in the ischemic tissue 6 h after occlusion [34–36]. NMR measurements of cerebral ischemic tissue extracts indicated that focal ischemia caused decrease in aspartate, glutamate, NAA and total creatine at 24 h after occlusion [34]. However, these studies were fragmented in terms of the metabolisms and the holistic metabolic responses of cerebral tissues to focal brain ischemia remained to be fully understood.
Stroke is a leading cause of death and disability, affecting millions of people worldwide with almost 80% of them as ischemic stroke and understanding the multiple mechanisms underlying cerebral ischemia is essential for development of effective treatments. To understand metabolic changes induced by focal brain ischemia, we conducted a comparative analysis of metabolic composition of cerebral tissue from rats with sham-operation and middle cerebral artery occlusion (MCAO) using high-resolution nuclear magnetic resonance (NMR) spectroscopy. More than 40 metabolites were assigned including organic acids, amino acids, carbohydrates, choline, pyrimidine and purine metabolites. Our results showed that MCAO led to significant level decreases for glutamate, glutamine, aspartate, γ-aminobutyrate (GABA), taurine, malate, fumarate, acetate, phosphocreatine, and purine and pyrimidine metabolites such as inosine, hypoxanthine, xanthine, uracil and UDP/UTP, together with significant level increases for glucose in focal brain tissue extracts. This demonstrated that experimental ischemic stroke in rats caused extensive perturbation in tricarboxylic acid cycle, GABA shunt, and metabolisms of choline and nucleic acids. These findings provided essential information for our understandings of MCAO-caused biochemical alterations and demonstrated the metabolite composition analysis as a useful tool for understanding the neurochemistry of stroke.
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Citation Excerpt :
Aspartate, glutamate, GABA, taurine, myo-inositol and phosphoethanolamine are those preferentially released, as documented in a multitude of reports (rev. in Phillis and O'Regan, 2003). A corresponding decrease in osmolyte brain levels reflects the magnitude of the efflux (Nonaka et al., 1998). Efflux of taurine, phosphoethanolamine and myo-inositol is predominantly caused by swelling (Phillis and O'Regan, 2003), whereas swelling is not the only stimulus for osmolytes such as GABA and glutamate which also serve as neurotransmitters.
Cerebral water control is critical to maintain neuronal excitability, and to prevent injuries derived from brain swelling or shrinkage. The influence of aquaporins (AQPs) in the balance of water distribution between intracranial compartments is getting much experimental support. The importance of AQPs in fluid clearance during vasogenic brain edema seems well established but their role in cytotoxic swelling and in brain cell shrinkage is not known in detail. The main AQPs function as water channels anticipates their influence on cell volume changes as well as on the mechanisms of volume recovery, which include notably the osmolyte translocation across the cell membrane. Osmolyte fluxes permit the reestablishment of an osmotic balance and volume recovery in anisosmotic-elicited cell volume changes, but are also causal factors per se of brain cell swelling or shrinkage in pathological situations. This review aims to inform on the so far described functional interactions between AQPs and osmolyte fluxes and their volume-sensitive pathways. It also points to the coincidence of AQPs and activation of osmolyte fluxes in physiological and pathological conditions and to the importance of finding possible functional links between these two events, thus enlarging the possibilities via AQP manipulations, to prevent the adverse consequences of cell volume changes in brain.

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Journal of the Neurological Sciences

Abstract

Introduction

Section snippets

Focal ischemia model

Results

Discussion

References (25)

¹H-NMR detection of cerebral myo-inositol

FEBS lett

Magnetic resonance spectroscopy of multiple sclerosis: in vivo detection of myelin breakdown products

Lancet

Hyponatremia causes large sustained reductions in brain content of multiple organic osmolytes in rats

Brain Res.

Blood-brain barrier permeability and brain concentration of sodium, potassium, and chloride during focal ischemia

J. Cereb. Blood Flow Metab.

Multiple sclerosis in children: cerebral metabolic alterations monitored by localized proton magnetic resonance spectroscopy

Ann. Neurol.

Control of brain volume during hyperosmolar and hypoosmolar conditions

Ann. Rev. Med.

Ischemia-induced shift of inhibitory and excitatory amino acids from intra- to extracellular compartments

J. Cereb. Blood Flow Metab.

Characterization of the major brain osmolytes that accumulate in salt-loaded rats

Am. J. Physiol.

Experimental studies of ischemic brain edema: 1. A new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area

Jpn. J. Stroke

Role of organic osmolytes in myelinolysis. A topographic study in rats after rapid correction of hyponatremia

J. Clin. Invest.

Elevation of neuroactive substances in the cortex of cats during prolonged focal ischemia

J. Cereb. Blood Flow Metab.

Contributions of ions and albumin to the formation and resolution of ischemic brain edema

J. Neurosurg.

Cited by (28)

Osmolytes: Wonder molecules to combat protein misfolding against stress conditions

Neurochemical changes in unilateral cerebral hemisphere during the subacute stage of focal cerebral ischemia-reperfusion in rats: An ex vivo <sup>1</sup> H magnetic resonance spectroscopy study

Rapamycin ameliorates brain metabolites alterations after transient focal ischemia in rats

Increased brain lactate is central to the development of brain edema in rats with chronic liver disease

NMR analysis of the rat neurochemical changes induced by middle cerebral artery occlusion

Brain volume regulation: Osmolytes and aquaporin perspectives