Cytoskeleton Elements Contribute to Prion Peptide-Induced Endothelial Barrier Breakdown in a Blood-Brain Barrier In Vitro System.

The mechanisms involved in the interaction of PrP 106-126, a peptide corresponding to the prion protein amyloidogenic region, with the blood-brain barrier (BBB) were studied. PrP 106-126 treatment that was previously shown to impair BBB function, reduced cAMP levels in cultured brain endothelial cells, increased nitric oxide (NO) levels, and changed the activation mode of the small GTPases Rac1 (inactivation) and RhoA (activation). The latter are well established regulators of endothelial barrier properties that act via cytoskeletal elements. Indeed, liquid chromatography-mass spectrometry (LC-MS)-based proteomic profiling study revealed extensive changes in expression of cytoskeleton-related proteins. These results shed light on the nature of the interaction between the prion peptide PrP 106-126 and the BBB and emphasize the importance of the cytoskeleton in endothelium response to prion- induced stress.

Combined Treatment of an Amyotrophic Lateral Sclerosis Rat Model with Recombinant GOT1 and Oxaloacetic Acid: A Novel Neuroprotective Treatment.

BACKGROUND/AIM: The sporadic form of the disease affects the majority of amyotrophic lateral sclerosis (ALS) patients. The role of glutamate (Glu) excitotoxicity in ALS has been extensively documented and remains one of the prominent hypotheses of ALS pathogenesis. In light of this evidence, the availability of a method to remove excess Glu from brain and spinal cord extracellular fluids without the need to deliver drugs across the blood-brain barrier and with minimal or no adverse effects may provide a major therapeutic asset, which is the primary aim of this study. METHODS: The therapeutic efficacy of the combined treatment with recombinant Glu-oxaloacetate-transaminase (rGOT) and its co-factor oxaloacetic acid (OxAc) has been tested in an animal model of sporadic ALS. RESULTS: We found that OxAc/rGOT treatment provides significant neuroprotection to spinal cord motor neurons. It also slows down the development of motor weakness and prolongs survival. CONCLUSION: In this study we bring evidence that the administration of Glu scavengers to rats with sporadic ALS inhibited the massive death of spinal cord motor neurons, slowed the onset of motor weakness and prolonged survival. This treatment may be of high clinical significance for the future treatment of chronic neurodegenerative diseases.

Peptide derived from HIV-1 TAT protein destabilizes a monolayer of endothelial cells in an in vitro model of the blood-brain barrier and allows permeation of high molecular weight proteins.

Most chemotherapeutic agents are blood-brain barrier (BBB) impermeants. HIV-1-derived TAT protein variants contain a transmembrane domain, which may enable them to cross the BBB and reach the brain. Here we synthesized CAYGRKKRRQRRR, a peptide containing a cysteine moiety attached to the N terminus of the transmembrane domain (C-TAT peptide), and studied its effects in an in vitro BBB model, which we found to reflect penetration by a receptor-independent pathway. Incubation of the brain capillary endothelial cell monolayer with 0.3-0.6 µmol/ml of this C-TAT peptide, for a period of 1-2 h, destabilizes brain capillary endothelial cell monolayer and introduces the ability of impermeant therapeutic agents including high molecular weight proteins to penetrate it substantially. The cysteinyl moiety at position 1 of the C-TAT peptide contributes largely to the destabilizing potency and the penetration efficacy of impermeant substances. The destabilizing effect was reversed using heparin. In summary, experimental conditions allowing a significant increase in entry of impermeant low and high molecular weight substances from the luminal (blood) to the abluminal side (brain) were found in an in vitro BBB model reflecting in vivo protein penetrability by a receptor-independent pathway.

Blood glutamate scavengers prolong the survival of rats and mice with brain-implanted gliomas.

L-Glutamate (Glu) plays a crucial role in the growth of malignant gliomas. We have established the feasibility of accelerating a naturally occurring brain to-blood Glu efflux by decreasing blood Glu levels with intravenous oxaloacetate, the respective Glu co-substrate of the blood resident enzyme humane glutamate­oxaloacetate transaminase(hGOT). We wished to demonstrate that blood Glu scavenging provides neuroprotection in the case of glioma.We now describe the neuroprotective effects of blood Glu scavenging in a fatal condition such as brain-implanted C6 glioma in rats and brain-implanted human U87 MG glioma in nude mice. Rat (C-6) or human (U87) glioma cells were grafted stereotactically in the brain of rats or mice. After development of tumors, the animals were drinking oxaloacetate with or without injections of hGOT. In addition, mice were treated with combination treatment, which included drinking oxaloacetate with intracutaneous injections of hGOT and intraperitoneal injection of Temozolomide. Animals drinking oxaloacetate with or without injections of hGOT displayed a smaller tumor volume, reduced invasiveness and prolonged survival than control animals drinking saline. These effects were significantly enhanced by Temozolomide in mice, which increased survival by 237%. This is the first demonstration of blood Glu scavenging in brain cancer, and because of its safety, is likely to be of clinical significance for the future treatment of human gliomas. As we demonstrated, the blood glutamate scavenging treatment in combination with TMZ could be a good candidate or as an alternative treatment to the patients that do not respond to TMZ.

Blood glutamate scavengers prolong the survival of rats and mice with brain-implanted gliomas.

L-Glutamate (Glu) plays a crucial role in the growth of malignant gliomas. We have established the feasibility of accelerating a naturally occurring brain to-blood Glu efflux by decreasing blood Glu levels with intravenous oxaloacetate, the respective Glu co-substrate of the blood resident enzyme humane glutamate-oxaloacetate transaminase (hGOT). We wished to demonstrate that blood Glu scavenging provides neuroprotection in the case of glioma. We now describe the neuroprotective effects of blood Glu scavenging in a fatal condition such as brain-implanted C6 glioma in rats and brain-implanted human U87 MG glioma in nude mice. Rat (C-6) or human (U87) glioma cells were grafted stereotactically in the brain of rats or mice. After development of tumors, the animals were drinking oxaloacetate with or without injections of hGOT. In addition, mice were treated with combination treatment, which included drinking oxaloacetate with intracutaneous injections of hGOT and intraperitoneal injection of Temozolomide. Animals drinking oxaloacetate with or without injections of hGOT displayed a smaller tumor volume, reduced invasiveness and prolonged survival than control animals drinking saline. These effects were significantly enhanced by Temozolomide in mice, which increased survival by 237%. This is the first demonstration of blood Glu scavenging in brain cancer, and because of its safety, is likely to be of clinical significance for the future treatment of human gliomas. As we demonstrated, the blood glutamate scavenging treatment in combination with TMZ could be a good candidate or as an alternative treatment to the patients that do not respond to TMZ.

Effect of glutamate and blood glutamate scavengers oxaloacetate and pyruvate on neurological outcome and pathohistology of the hippocampus after traumatic brain injury in rats.

BACKGROUND: Decreasing blood glutamate concentrations after traumatic brain injury accelerates brain-to-blood glutamate efflux, leading to improved neurologic outcomes. The authors hypothesize that treatment with blood glutamate scavengers should reduce neuronal cell loss, whereas administration of glutamate should worsen outcomes. The authors performed histologic studies of neuronal survival in the rat hippocampus after traumatic brain injury and treatment with blood glutamate scavengers. METHODS: Traumatic brain injury was induced on anesthetized male Sprague-Dawley rats by a standardized weight drop. Intravenous treatment groups included saline (control), oxaloacetate, pyruvate, and glutamate. Neurologic outcome was assessed using a Neurological Severity Score at 1 h, and 1, 2, 7, 14, 21, 28 days. Blood glutamate was determined at baseline and 90 min. Four weeks after traumatic brain injury, a histologic analysis of surviving neurons was performed. RESULTS: Oxaloacetate and pyruvate treatment groups demonstrated increased neuronal survival (oxaloacetate 2,200 ± 37, pyruvate 2,108 ± 137 vs. control 1,978 ± 46, P < 0.001, mean ± SD). Glutamate treatment revealed decreased neuronal survival (1,715 ± 48, P < 0.001). Treatment groups demonstrated favorable neurologic outcomes at 24 and 48 h (Neurological Severity Score at 24 and 48 h: 5.5 (1-8.25), 5 (1.75-7.25), P = 0.02 and 3(1-6.5), 4 (1.75-4.5), P = 0.027, median ± corresponding interquartile range). Blood glutamate concentrations were decreased in the oxaloacetate and pyruvate treatment groups. Administration of oxaloacetate and pyruvate was not shown to have any adverse effects. CONCLUSIONS: The authors demonstrate that the blood glutamate scavengers oxaloacetate and pyruvate provide neuroprotection after traumatic brain injury, expressed both by reduced neuronal loss in the hippocampus and improved neurologic outcomes. The findings of this study may bring about new therapeutic possibilities in a variety of clinical settings.

Interactions of the prion peptide (PrP 106-126) with brain capillary endothelial cells: coordinated cell killing and remodeling of intercellular junctions.

We studied here the interactions of PrP 106-126, a peptide corresponding to the prion protein (PrP) amyloidogenic region, with a blood-brain barrier in vitro model consisting of confluent porcine brain endothelial cells (PBEC). PrP 106-126 interacted selectively with PBEC via their luminal side, and caused cumulative cell death, as shown by lactate dehydrogenase release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction, Caspase 3 induction and direct cell counting. In addition, PrP 106-126, but not its corresponding scrambled peptide, produced a 50% reduction of the trans-endothelial electrical resistance, while the PBEC maintained confluency. This process was accompanied by a 23% increase of PBEC average size and the selective disappearance from the cell borders of the junction proteins occludin, claudin-5 and VE-cadherin (but not ZO-1), as evaluated by immunostaining. These results fit with a mechanism by which PrP 106-126 initiates a coordinated cell killing process ultimately causing the remaining cells to undergo a coordinated remodeling of the intercellular junctions and an expansion of their cell territory.

Pyruvate's blood glutamate scavenging activity contributes to the spectrum of its neuroprotective mechanisms in a rat model of stroke.

In previous studies, we have shown that by increasing the brain-to-blood glutamate efflux upon scavenging blood glutamate with either oxaloacetate or pyruvate, one achieves highly significant neuroprotection particularly in the context of traumatic brain injury. The current study examines, for the first time, how the blood glutamate scavenging properties of glutamate-pyruvate transaminase (GPT), alone or in combination with pyruvate, may contribute to the spectrum of its neuroprotective mechanisms and improve the outcome of rats exposed to brain ischemia, as they do after head trauma. Rats that were exposed to permanent middle cerebral artery occlusion (MCAO) and treated with intravenous 250 mg/kg pyruvate had a smaller volume of infarction and reduced brain edema, resulting in an improved neurological outcome and reduced mortality compared to control rats treated with saline. Intravenous pyruvate at the low dose of 31.3 mg/kg did not demonstrate any neuroprotection. However, when combined with 0.6 mg/kg of GPT there was a similar neuroprotection observed as seen with pyruvate at 250 mg/kg. Animals treated with 1.69 g/kg glutamate had a worse neurological outcome and a larger extent of brain edema. The decrease in mortality, infarcted brain volume and edema, as well as the improved neurological outcome following MCAO, was correlated with a decrease in blood glutamate levels. We therefore suggest that the blood glutamate scavenging activity of GPT and pyruvate contributes to the spectrum of their neuroprotective mechanisms and may serve as a new neuroprotective strategy for the treatment of ischemic stroke.

The effects of estrogen and progesterone on blood glutamate levels: evidence from changes of blood glutamate levels during the menstrual cycle in women.

The gonadal steroids estrogen and progesterone have been shown to have neuroprotective properties against various neurodegenerative conditions. Excessive concentrations of glutamate have been found to exert neurotoxic properties. We hypothesize that estrogen and progesterone provide neuroprotection by the autoregulation of blood and brain glutamate levels. Venous blood samples (10 ml) were taken from 31 men and 45 women to determine blood glutamate, estrogen, progesterone, glucose, glutamate-pyruvate transaminase (GPT), and glutamate-oxaloacetate transaminase (GOT) levels, collected on Days 1, 7, 12, and 21 of the female participants' menstrual cycle. Blood glutamate concentrations were higher in men than in women at the start of menstruation (P < 0.05). Blood glutamate levels in women decreased significantly on Days 7 (P < 0.01), 12 (P < 0.001), and 21 (P < 0.001) in comparison with blood glutamate levels on Day 1. There was a significant decrease in blood glutamate levels on Days 12 (P < 0.001) and 21 (P < 0.001) in comparison with blood glutamate levels on Day 7. Furthermore, there was an increase in blood glutamate levels on Day 21 compared with Day 12 (P < 0.05). In women, there were elevated levels of estrogen on Days 7 (P < 0.05), 12, and 21 (P < 0.001), and elevated levels of progesterone on Days 12 and 21 (P < 0.001). There were no differences between men and women with respect to blood glucose concentrations. Concentrations of GOT (P < 0.05) and GPT (P < 0.001) were significantly higher in men than in women during the entire cycle. The results of this study demonstrate that blood glutamate levels are inversely correlated to levels of plasma estrogen and progesterone.

The activation of ß2-adrenergic receptors in naïve rats causes a reduction of blood glutamate levels: relevance to stress and neuroprotection.

This study examines the effects of the activation of ß1 and ß2-adrenergic receptors on glutamate homeostasis in the blood of naïve rats. Forty five male Sprague-Dawley rats were randomly assigned into one of seven treatment groups that were treated with various ß-adrenergic receptor agonist and antagonist drugs. Blood glutamate levels were determined at t = 0, 30, 60, 90, and 120 min. The activation of ß1 and ß2-adrenergic receptors via isoproterenol hydrochloride administration produced a marked sustained decrease in blood glutamate levels by 60 min after treatment (ANOVA, t = 60, 90 min: P < 0.05, t = 120 min: P < 0.01). Pretreatment with propranolol hydrochloride (a non-selective ß-adrenergic receptor blocker) or butaxamine hydrochloride (a selective ß2-adrenergic receptor blocker) occluded the isoproterenol-mediated decrease in blood glutamate levels. Propranolol alone had no effect on blood glutamate levels. Selective ß1-adrenergic receptor blockade with metoprolol resulted in decreased blood glutamate levels (ANOVA, t = 90 min: P < 0.05, t = 120 min: P < 0.01). Butaxamine hydrochloride alone resulted in a delayed-onset increase in glutamate levels (ANOVA, t = 120 min: P < 0.05). The results suggest that the activation of ß2 receptors plays an important role in the homeostasis of glutamate in rat blood.

Distribution of radiolabeled l-glutamate and d-aspartate from blood into peripheral tissues in naive rats: significance for brain neuroprotection.

Excess l-glutamate (glutamate) levels in brain interstitial and cerebrospinal fluids (ISF and CSF, respectively) are the hallmark of several neurodegenerative conditions such as stroke, traumatic brain injury or amyotrophic lateral sclerosis. Its removal could prevent the glutamate excitotoxicity that causes long-lasting neurological deficits. As in previous studies, we have established the role of blood glutamate levels in brain neuroprotection, we have now investigated the contribution of the peripheral organs to the homeostasis of glutamate in blood. We have administered naive rats with intravenous injections of either l-[1-(14)C] Glutamic acid (l-[1-(14)C] Glu), l-[G-(3)H] Glutamic acid (l-[G-(3)H] Glu) or d-[2,3-(3)H] Aspartic acid (d-[2,3-(3)H] Asp), a non-metabolized analog of glutamate, and have followed their distribution into peripheral organs. We have observed that the decay of the radioactivity associated with l-[1-(14)C] Glu and l-[G-(3)H] Glu was faster than that associated with glutamate non-metabolized analog, d-[2,3-(3)H] Asp. l-[1-(14)C] Glu was subjected in blood to a rapid decarboxylation with the loss of (14)CO(2). The three major sequestrating organs, serving as depots for the eliminated glutamate and/or its metabolites were skeletal muscle, liver and gut, contributing together 92% or 87% of total l-[U-(14)C] Glu or d-[2,3-(3)H] Asp radioactivity capture. l-[U-(14)C] Glu and d-[2,3-(3)H] Asp showed a different organ sequestration pattern. We conclude that glutamate is rapidly eliminated from the blood into peripheral tissues, mainly in non-metabolized form. The liver plays a central role in glutamate metabolism and serves as an origin for glutamate metabolites that redistribute into skeletal muscle and gut. The findings of this study suggest now that pharmacological manipulations that reduce the liver glutamate release rate or cause a boosting of the skeletal muscle glutamate pumping rate are likely to cause brain neuroprotection.

Effects of blood glutamate scavenging on cortical evoked potentials.

It is well known that traumatic or ischemic brain injury is followed by acute excitotoxicity caused by the presence of abnormally high glutamate (Glu) in brain fluids. It has recently been demonstrated that excess Glu can be eliminated from brain into blood following the intravenous administration of oxaloacetate (OxAc), which, by scavenging blood Glu, induces an enhanced and neuroprotective brain-to-blood Glu efflux. In this study, we subjected rats to intravenous OxAc administration (i.v., 12.5, 25, and 50 mg/kg, respectively), and studied its effects on somatosensory evoked cortical potentials (EPs). Against our expectation, the amplitudes of EPs did not decrease but increased in a dose- and time-dependent manner after OxAc administration. Similar effects were observed when blood Glu scavenging was enhanced by combining OxAc (12.5 mg/kgbw) with recombinant glutamate-oxaloacetate transaminase (GOT, 0.14 nmol/100 g rat). On the basis of these results, we suggest that the changes of amplitudes of the EPs involve not only a glutamatergic but also the weakening of a GABAergic component. We cannot rule out the possibility that OxAc penetrates into the brain and improves mitochondrial functions.

The effect of hyperthermia on blood glutamate levels.

INTRODUCTION: Glutamate neurotoxicity is determined by the balance between glutamate release within the brain and efflux of excess glutamate from the brain. Brain-to-blood efflux of glutamate is increased by decreasing the concentration of glutamate in blood. Little is known about the effect of hyperthermia on blood glutamate concentrations, and the effectiveness of blood glutamate-decreasing mechanisms in these conditions. Although hyperthermia is hypothesized to decrease blood glutamate concentrations by activation of stress mechanisms, blunting the stress response by blocking ß-adrenergic receptors should prevent this decrease. Furthermore, during hyperthermia there should be a concurrent process of leakage of glutamate from muscle tissue into blood, resulting in a contradictory increase of blood glutamate concentrations. In this study we investigated the effects of hyperthermia on blood glutamate levels and studied the effects of the ß-adrenergic receptor antagonist propranolol on stress-induced changes in glutamate levels. We then studied the effectiveness of the blood glutamate scavenger oxaloacetate on hyperthermia-induced increases of glutamate levels. MATERIALS AND METHODS: Twenty-four rats were randomly divided into 3 groups. Rats' body temperatures were increased (by 1°C every 40 minutes) from 37°C to 42°C. The first group received 1 mL per 100 g of isotonic saline (control). The second group received 1 mL per 100 g of 1M oxaloacetate when the temperature reached 39°C. The third group received 10 mg/kg of propranolol before initiation of the warming. RESULTS: Warming the rats from 37°C to 39°C decreased the blood glutamate levels in the control group (P < 0.01) and oxaloacetate treatment group (P < 0.0001), whereas further increases in temperature from 40°C to 42°C increased the blood glutamate levels (P < 0.01 and P < 0.0001, respectively). Pretreatment with propranolol prevented the decrease in blood glutamate concentrations seen in mild hyperthermia and did not affect the increase in blood glutamate levels seen at temperatures of 41°C and 42°C (P < 0.005). DISCUSSION: The results of this study demonstrated that hyperthermia leads to decreases in glutamate levels in the blood, presumably by activation of the sympathetic nervous system. Oxaloacetate, previously reported to reduce blood glutamate levels at 37°C, was ineffective at temperatures over 40°C. Propranolol pretreatment blunted the initial decrease in blood glutamate, and thereafter had no effect when compared with control and treatment groups. Understanding the mechanisms underlying glutamate regulation in the blood during states of hyperthermia and stress has important clinical implications in treating neurodegenerative conditions.

Oxaloacetate decreases the infarct size and attenuates the reduction in evoked responses after photothrombotic focal ischemia in the rat cortex.

A traumatic brain injury or a focal brain lesion is followed by acute excitotoxicity caused by the presence of abnormally high glutamate (Glu) levels in the cerebrospinal and interstitial fluids. It has recently been demonstrated that this excess Glu in the brain can be eliminated into the blood following the intravenous administration of oxaloacetate (OxAc), which, by scavenging the blood Glu, induces an enhanced and neuroprotective brain-to-blood Glu efflux. In this study, we subjected rats to a photothrombotic lesion and treated them after the illumination with a single 30-min-long administration of OxAc (1.2 mg/100 g, i.v.). Following induction of the lesion, we measured the infarct size and the amplitudes of the somatosensory evoked potentials (SEPs) as recorded from the skull surface. The photothrombotic lesion resulted in appreciably decreased amplitudes of the evoked potentials, but OxAc administration significantly attenuated this reduction, and also the infarct size assessed histologically. We suggest that the neuroprotective effects of OxAc are due to its blood Glu-scavenging activity, which, by increasing the brain-to-blood Glu efflux, reduces the excess Glu responsible for the anatomical and functional correlates of the ischemia, as evaluated by electrophysiological evoked potential (EP) measurements.

Autoimmune epilepsy: distinct subpopulations of epilepsy patients harbor serum autoantibodies to either glutamate/AMPA receptor GluR3, glutamate/NMDA receptor subunit NR2A or double-stranded DNA.

We studied 82 patients with different types of epilepsy and 49 neurologically intact non-epileptic controls, and identified three different subpopulations of epilepsy patients bearing significantly elevated levels of autoantibodies to either GluR3B-peptide of glutamate/AMPA receptor subtype 3 (17/82; 21% of patients), or to a peptide of NR2A subunit of glutamate/NMDA receptors (15/82; 18%), or to double-stranded (ds) DNA, the hallmark of systemic lupus erythematosus (13/80; 16%). Most patients had only one antibody type, arguing against cross-reactivity. Nearly all anti-dsDNA Ab-positive patients did not harbor anti-nuclear autoantibodies. Most patients had no history of brain damage, febrile convulsions, early onset epilepsy, acute epilepsy or intractable seizures. We suggest to measure the 'autoimmune-fingerprints' of epilepsy patients for diagnostic and therapeutic purposes.

Blood glutamate scavenging as a novel neuroprotective treatment for paraoxon intoxication.

Organophosphate-induced brain damage is an irreversible neuronal injury, likely because there is no pharmacological treatment to prevent or block secondary damage processes. The presence of free glutamate (Glu) in the brain has a substantial role in the propagation and maintenance of organophosphate-induced seizures, thus contributing to the secondary brain damage. This report describes for the first time the ability of blood glutamate scavengers (BGS) oxaloacetic acid in combination with glutamate oxaloacetate transaminase to reduce the neuronal damage in an animal model of paraoxon (PO) intoxication. Our method causes a rapid decrease of blood Glu levels and creates a gradient that leads to the efflux of the excess brain Glu into the blood, thus reducing neurotoxicity. We demonstrated that BGS treatment significantly prevented the peripheral benzodiazepine receptor (PBR) density elevation, after PO exposure. Furthermore, we showed that BGS was able to rescue neurons in the piriform cortex of the treated rats. In conclusion, these results suggest that treatment with BGS has a neuroprotective effect in the PO intoxication. This is the first time that this approach is used in PO intoxication and it may be of high clinical significance for the future treatment of the secondary neurologic damage post organophosphates exposure.

Mechanisms of glutamate efflux at the blood-brain barrier: involvement of glial cells.

At high concentrations, glutamate (Glu) exerts potent neurotoxic properties, leading to irreversible brain damages found in numerous neurological disorders. The accepted notion that Glu homeostasis in brain interstitial fluid is maintained primarily through the activity of Glu transporters present on glial cells does not take into account the possible contribution of endothelial cells constituting the blood-brain barrier (BBB) to this process. Here, we present evidence for the presence of the Glu transporters, excitatory amino-acid transporters (EAATs) 1 to 3, in porcine brain endothelial cells (PBECs) and show their participation in Glu uptake into PBECs. Moreover, transport of Glu across three in vitro models of the BBB is investigated for the first time, and evidence for Glu transport across the BBB in both directions is presented. Our results provide evidence that the BBB can function in the efflux mode to selectively remove Glu, via specific transporters, from the abluminal side (brain) into the luminal compartment (blood). Furthermore, we found that glial cells lining the BBB have an active role in the efflux process by taking up Glu and releasing it, through hemichannels, anion channels, and possibly the reversal of its EAATs, in close proximity to ECs, which in turn take up Glu and release it to the blood.

Cell-free DNA as a marker for prediction of brain damage in traumatic brain injury in rats.

Traumatic brain injury (TBI) is a major cause of morbidity and mortality, and early predictors of neurological outcomes are of great clinical importance. Cell free DNA (CFD), a biomarker used for the diagnosis and monitoring of several diseases, has been implicated as a possible prognostic indicator after TBI. The purpose of this study was to determine the pattern and timing of CFD levels after TBI, and whether a relationship exists between the level of CFD and brain edema and neurological outcomes. Thirty-nine Sprague-Dawley rats were randomly assigned to two groups: rats in group 1 (sham group) were anesthetized and had a scalp incision without TBI, and rats in group 2 were anesthetized and had a scalp incision with TBI, which was induced by using a weight drop model that causes diffuse brain injury. A neurological severity score (NSS) was assessed at 1, 24, and 48 h after TBI. CFD was measured via blood samples drawn at t=0 (baseline), 12, 24, 48, 72, and 120 h after TBI. At 48 h after TBI, brain edema was determined in a subgroup of 11 rats by calculating the difference between rats' wet and dry brain weight. The significance of comparisons between and within groups (CFD levels, brain water content, and NSS) were determined using the Kruskal-Wallis, Mann-Whitney and Student t test. The correlation between CFD levels and the NSS, as well as between CFD levels and the extent of brain edema, was calculated using the Spearman and Pearson tests, respectively. Compared with baseline levels, the CFD levels in rats subjected to TBI were significantly increased at 24 and 48 h after TBI (p<0.01 and p<0.05, respectively). A positive correlation was demonstrated between CFD levels 24 h following TBI and the extent of brain edema (r=0.63, p<0.05), as well as between CFD levels and the NSS (r=0.79, p<0.005). In this study, we demonstrated an increase in CFD levels after TBI, as well as a correlation between CFD levels and brain edema and NSS. CFD levels may provide a quick, reliable, and simple prognostic indicator of neurological outcome in animals after TBI. Its role in humans has not been clearly elucidated, but has potentially significant clinical implications.

Effect of estrogens on blood glutamate levels in relation to neurological outcome after TBI in male rats.

PURPOSE: Estrogen has been shown to possess neuroprotective properties both in vitro and in vivo. Traumatic brain injury (TBI) in ovulating females results in favorable neurological outcomes when compared to males with similar insults. The brain-to-blood glutamate gradient removes excess glutamate from brain extracellular fluids (ECF). Enhancing this gradient leads to improved neurological outcomes following TBI. In this study we investigate the effect of female gonadal steroids on blood glutamate levels and neurological outcomes. METHODS: Forty male Sprague-Dawley rats were assigned to one of five groups: (1) sham, (2) Premarin treatment, (3) TBI, (4) TBI + Premarin treatment, and (5) TBI + Premarin pretreatment. TBI was induced, and estrogen and glutamate levels were determined at 0, 60, 120, 135, and 150 min. Neurological recovery was evaluated using the Neurological Severity Score (NSS) at 1 h and reassessed at 24 h post TBI. RESULTS: Premarin treatment groups demonstrated a decline in blood glutamate levels by 60 min. This decline was found to be more pronounced in the TBI + Premarin group, which maintained the decline throughout the experiment. At 120 min, the difference between groups was most pronounced (TBI + Premarin 99 ± 36 µM/l vs. control 200 ± 46 µM/l, p < 0.01). Neurological recovery was significantly better in the Premarin treatment group (NSS at 24 h 6 ± 1 vs. control 11 ± 1). CONCLUSIONS: Premarin injected into male rats significantly decreases blood glutamate levels in rats suffering TBI. This decrease is associated with improved neurological outcomes, thus implicating the role of estrogen in neuroprotection.

Anatomical location of arterial and venous lines significantly affects motor performance in rats.

Several motor-function scales have been developed to assess neurological function in animal models of stroke, subarachnoid hemorrhage and closed head injury. We hypothesize that the location of arterial and venous catheters, even in the absence of brain injury, may impact rats' motor performance. Our study examined the effect of catheter location, rate of infection and the time required for catheter placement. We further describe an original technique of tail artery cannulation without exposure of the artery. Sixty-one rats were anesthetized and randomly assigned to one of seven groups, including no catheter, tail artery or artery + vein catheters, or femoral artery or artery + vein catheters. A neurological severity score (NSS) was determined at 1 h, 24 h and 48 h after surgical preparation or catheter placement. NSS at 1 h after placement of unilateral and bilateral femoral catheters was higher than the NSS observed at 1 h after placement of tail arterial and venous catheters (P < 0.01). The NSS also was higher at 24 h in the bilateral femoral catheter groups as compared with the tail catheter groups (P < 0.05). There were no differences in the NSS observed between the groups that had tail catheters and the sham group at 1 h, 24 h or 48 h. Infection rate at the site of catheter placement and the time required for catheter placement was also higher in the femoral catheter groups (P < 0.001). Thus, we propose that the line location may bias a study's results and lead to deceptive interpretations of neurological assessment following rat head injury. Compared to femoral vessels, tail blood vessels are preferable locations for lines placement.