Lysine and arginine reduce the effects of cerebral ischemic insults and inhibit glutamate-induced neuronal activity in rats

Abstract

Intravenous administration of arginine was shown to be protective against cerebral ischemic insults via nitric oxide production and possibly via additional mechanisms. The present study aimed at evaluating the neuroprotective effects of oral administration of lysine (a basic amino acid), arginine, and their combination on ischemic insults (cerebral edema and infarction) and hemispheric brain swelling induced by transient middle cerebral artery occlusion/reperfusion in rats. Magnetic resonance imaging and 2,3,5-triphenyltetrazolium chloride staining were performed 2 days after ischemia induction. In control animals, the major edematous areas were observed in the cerebral cortex and striatum. The volumes associated with cortical edema were significantly reduced by lysine (2.0 g/kg), arginine (0.6 g/kg), or their combined administration (0.6 g/kg each). Protective effects of these amino acids on infarction were comparable to the inhibitory effects on edema formation. Interestingly, these amino acids, even at low dose (0.6 g/kg), were effective to reduce hemispheric brain swelling. Additionally, the effects of in vivo microiontophoretic (juxtaneuronal) applications of these amino acids on glutamate-evoked neuronal activity in the ventromedial hypothalamus were investigated in awake rats. Glutamate-induced neuronal activity was robustly inhibited by microiontophoretic applications of lysine or arginine onto neuronal membranes. Taken together, our results demonstrate the neuroprotective effects of oral ingestion of lysine and arginine against ischemic insults (cerebral edema and infarction), especially in the cerebral cortex, and suggest that suppression of glutamate-induced neuronal activity might be the primary mechanism associated with these neuroprotective effects.

Keywords: arginine; cerebral edema; glutamate receptor; ischemia; lysine; magnetic resonance imaging; microiontophoretic application; middle cerebral artery occlusion.

Figure 1

Examples of five consecutive coronal brain images of MCAO rats administrated with (A) saline or (B) lysine as measured by T₂-weighted MRI. Brain areas with hyperintensity (i.e., increase in water content) was observed in the left hemispheres, which represent presence of edema. Note that oral administration of lysine (2.0 g/kg) suppressed cerebral edema, especially in the cerebral cortex. Lysine also reduced hemispheric brain swelling. Slice thickness, 1.2 mm.

Figure 2

Reduction of whole cerebral edema volume by oral administration of lysine (Lys) and/or arginine (Arg) in MCAO rats. Administration of lysine alone at high dose (2.0 g/kg), or combined administration of lysine and arginine at low doses (0.6 g/kg each) significantly suppressed whole cerebral edema volume. Numbers between parentheses represent number of experiments. *p< 0.05, significance compared to saline-treated control groups by ANOVA followed by Fisher's PLSD test.

Figure 3

Effects of lysine (Lys) and/or arginine (Arg) on cortical and striatal edema in MCAO rats. Note that significant reduction of edema volume was observed in the cerebral cortex but not in the striatum by administration of lysine (2.0 g/kg), arginine (0.6 g/kg), or combined administration of lysine and arginine. *p< 0.05, significance compared to the saline-treated control group by ANOVA followed by Fisher's PLSD test.

Figure 4

Rostrocaudal extension of cerebral edema. The value 0 represents the bregma position in the antero-posterior axis, with more anterior parts represented by positive values and more posterior parts represented by negative values. Note that the extent of cerebral edema appeared to be reduced in overall levels by administration of lysine (Lys), arginine (Arg), or their combination.

Figure 5

Reduction of hemispheric brain swelling by oral administration of lysine (Lys) and/or arginine (Arg) in MCAO rats. Administration of lysine alone (0.6 and 2.0 g/kg), arginine alone (0.6 g/kg), or combined administration of lysine and arginine at low doses (0.6 g/kg each) significantly suppressed hemispheric brain swelling. *p< 0.05 and **p< 0.01, significance compared to the saline-treated control group by ANOVA followed by Fisher's PLSD test.

Figure 6

Reduction of infarct area by oral administration of lysine (Lys) and/or arginine (Arg) in MCAO rats. Infarct area was measured with TTC staining. Note that administration of lysine alone at high dose (2.0 g/kg) or combined administration of lysine and arginine at low dose (0.6 g/kg) significantly suppressed infarct in consistent to the effects on cerebral edema as shown in Figure 2. *p< 0.05, significance compared to the saline-treated control group by ANOVA followed by Fisher's PLSD test.

Figure 7

Positive correlation between cerebral edema measured by MRI and infarct area measured by TTC staining in MCAO rats. Data from all experimental groups were combined for analysis. Y = 76.8 + 1.100X, r = 0.736, p< 0.0001.

Figure 8

An example of inhibitory responses to lysine and arginine applied microiontophoretically on glutamate-induced single unit activity in a rat VMH neuron. (A) The position of VMH in the rat brain. (B) A schematic representation of the multi-barreled glass micropipette with a recording electrode. (C) Suppression of glutamate-induced activity by lysine and arginine. Firing rate of this neuron was little at the basal level; therefore glutamate (−10 or −20 nA) was applied continuously in the background to evoke steady-state levels of firing activity. Microiontophoretic applications (45 s) of either lysine or arginine did inhibit this glutamate-induced neuronal activity whereas neither Na⁺ nor Cl⁻ modified it. Lower trace was the continuous recording of the upper trace. Note that inhibitory responses by lysine and arginine occurred rapidly while the recovery of inhibitory responses occurred slowly for 1–2 min after cessation of the application. Numerical values under the chemicals represent current strength (nA) applied for ejection of each chemical from the tip of multi-barreled micropipettes. The similar responses were repeatedly observed in other neurons. Ordinate: Neuronal discharge rate/s. Abscissa: time. Bars: period of microiontophoretic applications.