Arginine-enriched diet limits plasma and muscle glutamine depletion in head-injured rats.

OBJECTIVE: Injury is associated with a depletion in glutamine (GLN) pools, which may contribute to impairment of immune and nutritional statuses. Total parenteral nutrition enriched with arginine (ARG) is able to generate GLN in surgical patients. We hypothesized that this same concept may be applicable to enteral administration and could be extended to muscle GLN reserves. This study investigated the ability of an enteral formula enriched with ARG to restore the GLN pools in an experimental model of head injury. METHODS: Twenty-five male Sprague-Dawley rats were randomized into 4 groups: ad libitum access to food, head injury plus free access to nutrition, head injury plus standard enteral nutrition (Sondalis), and an immune-enhancing diet (IED). The two enteral diets were adjusted to be isocaloric (290 kcal.kg(-1).d(-1)) and isonitrogenous (3.29 g.kg(-1).d(-1)) and were delivered for 4 d (24 h/24 h). After sacrifice, plasma and muscle amino acids were determined. RESULTS: Head injury was associated with a large depletion of muscle and plasma GLN pools that were restored by IED administration but not by the standard diet. Moreover, the IED but not the standard diet improved or normalized ornithine and glutamate pools, suggesting that the modification of GLN pools is related to ARG administration. CONCLUSION: In our model of head injury, our IED, a diet without free GLN, is efficient in restoring the plasma and muscle pools of GLN, probably due to its high ARG content.

Autoradiographic analysis of mouse brain kinin B1 and B2 receptors after closed head trauma and ability of Anatibant mesylate to cross the blood-brain barrier.

The potent non-peptide B2 receptor (R) antagonist, Anatibant mesylate (Ms) (LF 16-0687 Ms), reduces brain edema and improves neurological function recovery in various focal and diffuse models of traumatic brain injury in rodents. In the present study, alteration of kinin B1 and B2R after closed head trauma (CHT) and in vivo binding properties of Anatibant Ms (3 mg/kg, s.c.) injected 30 min after CHT were studied in mice by autoradiography using the radioligands [125I]HPP-Hoe 140 (B2R), and [125I]HPP-des-Arg10-Hoe 140 (B1R). Whereas B1R is barely detected in most brain regions, B2R is extensively distributed, displaying the highest densities in the hindbrain. CHT was associated with a slight increase of B1R and a decrease of B2R (10-50%) in several brain regions. Anatibant Ms (Ki = 22 pM) displaced the B2R radioligand from its binding sites in several areas of the forebrain, basal ganglia and hindbrain. Displacement was achieved in 1 h and persisted at 4 h post-injection. The inhibition did not exceed 50% of the total specific binding in non-injured mice. After CHT, the displacement by Anatibant Ms was higher and almost complete in the cortex, caudate putamen, thalamus, hippocampus, medial geniculate nucleus, ventral tegmental area, and raphe. Evans blue extravasation in brain tissue at 4 h after CHT was abolished by Anatibant Ms. It appeared that Anatibant Ms penetrated into the brain in sufficient amounts, particularly after disruption of the blood-brain barrier, to account for its B2R-mediated neuro- and vascular protective effects. The diminished binding of B2R after CHT may reflect the occupancy or internalization of B2R following the endogenous production of bradykinin (BK).

Impairment of lymphocyte function in head-injured rats: effects of standard and immune-enhancing diets for enteral nutrition.

BACKGROUND: The metabolic response to head injury (HI) is characterized by a dysimmunity which may be a risk factor of a septic state. The use of immune enhancing diets (IEDs) could be a promising approach to improve immune functions. The aim of the study was to investigate the consequences of HI on lymphocyte function and to determine the effects of an enteral IED comparatively to a standard enteral nutrition. METHOD: A rat model of HI by fluid percussion was used. Twenty-five male Sprague-Dawley rats were randomized into 4 groups: rats receiving standard chow diet ad libitum (AL), rats sustaining HI and receiving standard chow diet and enteral saline (HI), rats receiving the enteral standard diet Sondalis HP (HIS), and rats receiving the IED Crucial (HIC). The two enteral diets were infused continuously during 4 days after the HI and were isocaloric, isonitrogenous and isovolumic. RESULTS: HI induced a thymus atrophy (HI vs. AL, P<0.05), and an impairment in lymphocyte CD25 receptor density responsiveness to stimulation. The IED blunted thymus atrophy and allowed to preserve the stimulation of blood and Peyer patches lymphocytes (HIC: Stimulated vs. Basal, P<0.05). CONCLUSION: IED seems more adapted for preserving lymphocyte function than standard diet in HI patients.

NMDA receptor activation inhibits alpha-secretase and promotes neuronal amyloid-beta production.

Acute brain injuries have been identified as a risk factor for developing Alzheimer's disease (AD). Because glutamate plays a pivotal role in these pathologies, we studied the influence of glutamate receptor activation on amyloid-beta (Abeta) production in primary cultures of cortical neurons. We found that sublethal NMDA receptor activation increased the production and secretion of Abeta. This effect was preceded by an increased expression of neuronal Kunitz protease inhibitory domain (KPI) containing amyloid-beta precursor protein (KPI-APP) followed by a shift from alpha-secretase to beta-secretase-mediated APP processing. This shift is a result of the inhibition of the alpha-secretase candidate tumor necrosis factor-alpha converting enzyme (TACE) when associated with neuronal KPI-APPs. This KPI-APP/TACE interaction was also present in AD brains. Thus, our findings reveal a cellular mechanism linking NMDA receptor activation to neuronal Abeta secretion. These results suggest that even mild deregulation of the glutamatergic neurotransmission may increase Abeta production and represent a causal risk factor for developing AD.

Fenofibrate, a peroxisome proliferator-activated receptor alpha agonist, exerts neuroprotective effects in traumatic brain injury.

Peroxisome proliferator-activated receptor alpha (PPARalpha) has been demonstrated to reduce inflammation in various inflammatory diseases. As traumatic brain injury (TBI) caused a neuroinflammatory response, we examined the effect of fenofibrate, a PPARalpha agonist, on the post-traumatic consequences caused by lateral fluid percussion of brain in rats. The effects of fenofibrate (50 and 100mg/kg) were evaluated on the consequences of TBI in the early phase (6 and 24h) and the late phase (7 days) after TBI. Neurological deficit, brain lesion, cerebral oedema and ICAM-1 expression were evaluated. Treatment with fenofibrate (given p.o. at 1 and 6h after TBI) decreases the neurological deficit induced by TBI at 24h. Furthermore, fenofibrate reduces brain oedema and ICAM-1 expression at 24h post-TBI. Rats given fenofibrate at 1, 6, 24, 48 and 72h after TBI show neurological recovery associated with a reduction of the brain lesion at 7 days post-TBI. The present data represents the first demonstration that fenofibrate, a PPARalpha agonist, exerts neuroprotective effects in TBI. The activation of receptor PPARalpha could be beneficial by counteracting the deleterious inflammatory response following TBI. This suggests that PPARalpha activation could be a new and promising therapeutic strategy for the treatment of brain trauma.

Beneficial effects of PJ34 and INO-1001, two novel water-soluble poly(ADP-ribose) polymerase inhibitors, on the consequences of traumatic brain injury in rat.

Traumatic brain injury produces peroxynitrite, a powerful oxidant which triggers DNA strand breaks, leading to the activation of poly(ADP-ribose)polymerase-1 (PARP-1). We previously demonstrated that 3-aminobenzamide, a PARP inhibitor, is neuroprotective in a model of traumatic brain injury induced by fluid percussion in rat, suggesting that PARP-1 could be a therapeutic target. In order to confirm this hypothesis, we investigated the effects of PJ34 and INO-1001, two PARP inhibitors from structural classes other than benzamide, on the post-traumatic consequences. Pre- and post-treatments with PJ34 (30 mg/kg/day) and INO-1001 (10 mg/kg/day) decrease the neurological deficit at 3 days post-injury and this deficit is still reduced at 7 days. These neurological recovery-promoting effects are associated with the inhibition of PARP-1 activation caused by trauma, as demonstrated by abolishment of immunostaining of poly(ADP-ribose). Thus, the present work strengthens strongly the concept that PARP-1 inhibition may be a suitable approach for the treatment of brain trauma.

Characterization of the alteration of nutritional state in brain injury induced by fluid percussion in rats.

OBJECTIVE: Patients suffering from traumatic brain injury (TBI) undergo rapid weight loss with negative nitrogen balance and enhanced whole-body protein breakdown, with protein wasting causing morbidity and increased mortality. Many experimental models of TBI have been used to evaluate strategies to improve the outcome of these patients, but nutritional status has not been considered in experiments published to date, although this may have great importance and influence the results obtained with TBI models. This study characterized the hypercatabolism level and nutritional status of TBI rats. DESIGN: Twenty-four male Wistar rats were randomized into three groups. Rats from the TBI group were anesthetized and fluid percussion was applied. The pair-fed (PF) group was healthy but was pair-fed to the TBI group. The ad libitum (AL) group was healthy and fed ad libitum. The study was performed over 10 days post-TBI. MEASUREMENTS AND RESULTS: TBI in rats was characterized by remarkable long-lasting anorexia, renal failure (creatinine clearance: AL 1.8+/-0.2 and PF 1.5+/-0.1 vs. TBI 0.9+/-0.1 l/24 hour), anorexia (appetite depressed throughout the study), increased myofibrillar proteolysis (3-methylhistidine/creatinine ratio (day 2: AL 36+/-1 and PF 38+/-2 vs. TBI 54+/-5 micromol/mmol), and intestinal atrophy (ileum: AL 29.3+/-2.5 and PF 28.7+/-1.1 vs. TBI 22.5+/-1.4 mg/cm). In addition, anorexia led to muscular atrophy and decreased nitrogen balance. The metabolic alterations described above can increase morbidity and mortality. CONCLUSIONS: TBI by fluid percussion in rats is a model reproducing the metabolic and nutritional alterations observed in clinical practice and is suitable for further studies exploring the efficacy of optimized nutritional support.

Cortical calcium increase following traumatic brain injury represents a pitfall in the evaluation of Ca2+-independent NOS activity.

In this report, our findings highlighted the presence of a high level of calcium in the cortex following traumatic brain injury (TBI) in a rat model of fluid percussion-induced brain injury. This calcium increase represents a pitfall in the assessment of Ca2+-independent nitric oxide synthase (NOS) activity supposed to play a role in the secondary brain lesion following TBI. The so-called Ca2+-independent NOS activity measured in the injured cortex 72 h after TBI had the pharmacological profile of a Ca2+-dependent NOS and was therefore inhibited with a supplement of calcium chelator. The remaining activity was very low and iNOS protein was hardly immunodetected on the same sample used for NOS activity assay. The concentration of calcium chelator used in the assay should be revised and adjusted consequently to make sure that the calcium-free condition is achieved for the assay. Otherwise, the findings tend towards an overestimation of Ca2+-independent and underestimation of Ca2+-dependent NOS activities. The revised Ca2+-independent NOS activity assay was then tested, in relation with the amount of iNOS protein, in a model of LPS-induced neuroinflammation. Taken together, precautions should be taken when assessing the Ca2+-independent enzymatic activity in cerebral tissue after a brain insult.

Deleterious poly(ADP-ribose)polymerase-1 pathway activation in traumatic brain injury in rat.

Traumatic brain injury produces nitric oxide and reactive oxygen species. Peroxynitrite, resulting from the combination of nitric oxide and superoxide anions, triggers DNA strand breaks, leading to the activation of poly(ADP-ribose)polymerase-1. As excessive activation of this enzyme induces cell death, we examined the production of nitrosative stress, the activation of poly(ADP-ribose)polymerase-1, and the role of this enzyme in the outcomes of traumatic brain injury produced by fluid percussion in rats. Immunohistochemistry showed that 3-nitrotyrosine, an indicator of nitrosative stress, and poly(ADP-ribose), a marker of poly(ADP-ribose)polymerase-1 activation, were present as early as 30 min post-injury, and that persisted for 72 h. The poly(ADP-ribose)polymerase inhibitor, 3-aminobenzamide, at 10 and 30 mg/kg, significantly improved the neurological deficit, with a 60% reduction in the brain lesion volume and inhibition of poly(ADP-ribose)polymerase-1 activation. Thus, poly(ADP-ribose)polymerase-1 is involved in the neurological consequences of traumatic brain injury and may be a promising therapeutic target in clinical treatment of acute brain trauma.

LF 16-0687 Ms, a bradykinin B2 receptor antagonist, reduces ischemic brain injury in a murine model of transient focal cerebral ischemia.

1. Bradykinin promotes neuronal damage and brain edema through the activation of the B(2) receptor. The neuroprotective effect of LF 16-0687 Ms, a B(2) receptor antagonist, has been described when given prior to induction of transient focal cerebral ischemia in rat, but there are no data regarding the consequence of a treatment when given after injury. Therefore, in a murine model of transient middle cerebral artery occlusion (MCAO), we evaluated the effect of LF 16-0687 Ms given prior to and/or after the onset of ischemia on neurological deficit, infarct volume and inflammatory responses including cerebral edema, blood-brain barrier (BBB) disruption and neutrophil accumulation. 2. LF 16-0687 Ms (1, 2 and 4 mg kg(-1)) administered 0.5 h before and, 1.25 and 6 h after MCAO, decreased the infarct volume by a maximum of 33% and significantly improved the neurological recovery. 3. When given at 0.25 and 6.25 h after MCAO, LF 16-0687 Ms (1.5, 3 and 6 mg kg(-1)) decreased the infarct volume by a maximum of 25% and improved the neurological score. 4. Post-treatment with LF 16-0687 Ms (1.5 mg kg(-1)) significantly decreased brain edema (-28%), BBB disruption (-60%) and neutrophil accumulation (-65%) induced by ischemia. Physiological parameters were not modified by LF 16-0687 Ms. 5. These data emphasize the role of bradykinin B(2) receptor in the development of infarct lesion, neurological deficit and inflammatory responses resulting from transient focal cerebral ischemia. Therefore, B(2) receptor antagonist might represent a new therapeutic approach in the pharmacological treatment of stroke.

Neuroprotective effects of (S)-N-[4-[4-[(3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)carbonyl]-1-piperazinyl]phenyl]-2-thiophenecarboximid-amide (BN 80933), an inhibitor of neuronal nitric-oxide synthase and an antioxidant, in model of transient focal cerebral ischemia in mice.

Nitric oxide (NO) and reactive oxygen species are both implicated in neuronal death due to cerebral ischemia. BN 80933, an original compound associating an inhibitor of neuronal NO synthase with an antioxidant, has been shown to reduce functional and histological damage in rat submitted to cerebral ischemia. The aim of the present study was to confirm these results in mice and to further examine the effects of BN 80933 on inflammatory response, including blood-brain barrier (BBB) disruption, brain edema, and neutrophil infiltration after transient middle cerebral artery occlusion (MCAO). Intravenous administration of BN 80933 at 3 and 10 mg/kg 3 h after MCAO significantly reduced by 26 to 36% the infarct volume evaluated 24 and 48 h after ischemia, and improved the neurological score. Furthermore, BN 80933 at both dosages decreased by 42 to 75% the extravasation of Evans blue in brain parenchyma observed 24 h after ischemia. This reduction in BBB disruption was associated with decreased brain edema as demonstrated by the 37% reduction in brain water content induced by BN 80933 at 3 mg/kg 24 h after MCAO. Neutrophil infiltration in brain parenchyma, evaluated by the myeloperoxidase activity, was also reduced by 45 to 56% in animals treated with BN 80933 at 3 and 10 mg/kg. Together, these results extend the protective capacity of BN 80933 against brain ischemic injury and confirm that BN 80933 represents a promising treatment for stroke.

Deleterious activation of poly(ADP-ribose)polymerase-1 in brain after in vivo oxidative stress.

Oxidative stress has been shown to be implicated in the pathogenesis of central nervous system injuries such as cerebral ischemia and trauma, and chronic neurodegenerative diseases. In vitro studies show that oxidative stress, particularly peroxynitrite, could trigger DNA strand breaks, which lead to the activation of repairing enzymes including Poly(ADP-ribose) Polymerase-1 (PARP-1). As excessive activation of this enzyme induces cell death, we examined whether such a cascade also occurs in vivo in a model of oxidative stress in rat brain. For this purpose, the mitochondrial toxin malonate, which promotes free radical production, was infused into the left striatum of rats. Immunohistochemistry showed that 3-nitrotyrosine, an indicator of nitrosative stress, and poly(ADP-ribose), a marker of poly(ADP-ribose)polymerase-1 activation, were present as early as 1 h after malonate, and that they persisted for 24 h. The PARP inhibitor, 3-aminobenzamide, significantly reduced the lesion and inhibited PARP-1 activation induced by malonate. These results demonstrate that oxidative stress induced in vivo in the central nervous system leads to the activation of poly(ADP-ribose)polymerase-1, which contributes to neuronal cell death.

L-NAME reduces infarction, neurological deficit and blood-brain barrier disruption following cerebral ischemia in mice.

The role of nitric oxide (NO) in the development of post-ischemic cerebral infarction has been extensively examined, but fewer studies have investigated its role in other outcomes. In the present study, we first determined the temporal evolution of infarct volume, NO production, neurological deficit and blood-brain barrier disruption in a model of transient focal cerebral ischemia in mice. We then examined the effect of the nonselective NO-synthase inhibitor N(omega)-nitro-L-arginine-methylester (L-NAME). L-NAME given at 3 mg/kg 3 h after ischemia reduced by 20% the infarct volume and abolished the increase in brain NO production evaluated by its metabolites (nitrites/nitrates) 48 h after ischemia. L-NAME with this protocol also reduced the neurological deficit evaluated by the grip test and decreased by 65% the extravasation of Evans blue, an index of blood-brain barrier breakdown. These protective activities of L-NAME suggest that NO has multiple deleterious effects in cerebral ischemia.