Ann C. Rice

Repeated mild brain injuries result in cognitive impairment in B6C3F1 mice

Experimental investigations of single mild brain injury (SMI) show relatively little resultant cognitive impairment. However, repeated mild brain injuries (RMI), as those sustained by athletes (e.g., football, hockey, and soccer players) may have cumulative effects on cognitive performance and neuropathology. Numerous clinical studies show persistent, latent, and long-term consequences of RMI, unlike the episodic nature of SMI. The nature of repeated traumatic brain injury (TBI) introduces confounding factors in invasive and even semiinvasive animal models of brain injury (e.g., scar formation). Thus, the present study characterizes SMI and RMI in a noninvasive mouse weight drop model and the cumulative effects of RMI on cognitive performance. Investigation of drop masses and drop distances revealed masses of 50, 100, and 150 g dropped from 40 cm resulted in 0% mortality, no skull fracture, and no difference in acute neurological assessment following sham injury, SMI, or RMI. Cumulative effects of RMI were examined following four mild brain injuries 24 h apart induced by 50-, 100-, or 150-g masses dropped from 40 cm through histological measures, mean arterial pressure, and measures of complex/spatial learning. RMI produced no overt cell death within the cortex or hippocampus, no evidence of blood-brain barrier compromise, and no significant change in mean arterial pressure. Following testing in the Morris water maze (MWM) on days 7-11 after initial injury, mice in the RMI 100-g and RMI 150-g groups had significantly longer MWM goal latencies compared to sham, SMI 150-g, and RMI 50-groups. Additionally, the evident cognitive deficit manifested in the absence of observed cell death. This is the first study to show complex/spatial learning deficits following RMI, similar to the visual/spatial perception and planning deficits observed in clinical studies.

Status Epilepticus Causes Long‐Term NMDA Receptor‐Dependent Behavioral Changes and Cognitive Deficits

Summary: Purpose: The role of N‐methyl‐d‐aspartate (NMDA)‐receptor activation on behavioral and cognitive changes after status epilepticus (SE) is unknown. In this study, behavioral and cognitive changes after SE were evaluated in the short and long term and in rats in which the NMDA receptor was inactivated during SE.

Effect of lactate therapy upon cognitive deficits after traumatic brain injury in the rat.

SummaryBackground. In previous studies, it has been shown that intravenous lactate therapy can improve brain neurochemistry, adenosine triphosphate (ATP) generation and outcome after traumatic brain injury (TBI) in rats. In this study, we examined: (1) four L-lactate concentrations to determine the optimal therapeutic dose post TBI in terms of cognitive function; (2) ATP production after TBI for the L-lactate concentration found to be the optimal dose; (3) the possible production of lactic acidosis with the highest L-lactate concentration tested. Methods. Thirty minutes following a fluid percussion injury (FPI) over the left cerebral hemisphere, the animals received an intravenous infusion of 10, 28, 100, or 280 mM L-lactate (n = 10 for each group) for 3 h at a rate of 0.65 ml/h. Shams and control injured animals received a saline infusion. At 11–15 days post injury, cognitive deficits were examined using the Morris Water Maze (MWM) test. Three groups of rats were used for ATP analysis: shams, injured + saline infusion, and injury + the optimal lactate dose as determined by the MWM (n = 4/group). Additionally, a group receiving 280 mM L-lactate (n = 5) and one receiving a saline infusion (n = 3) were monitored for arterial blood variables and blood pressures. Findings. In the MWM test, only the 100 mM L-lactate-treated injured animals showed a significant reduction in cognitive deficits when compared to saline-treated injured animals (p ≤ 0.05). In the ATP study, injured animals without treatment had a 53% reduction in ATP level in the ipsilateral cortex, while animals with 100 mM lactate treatment had a 28% reduction. (p ≤ 0.05). No lactic acidosis was induced by the intravenous infusion of 280 mM L-lactate. Conclusions. This study indicates that the intravenous infusion of 100 mM L-lactate provided the optimal concentration of the substrate to ameliorate cognitive impairment, probably via the regeneration of ATP following TBI in rats.

Intraventricular Infusion of the Neurotrophic Protein S100B Improves Cognitive Recovery after Fluid Percussion Injury in the Rat

Elevated serum S100B levels have been shown to be a predictor of poor outcome after traumatic brain injury (TBI). Experimental data, on the other hand, demonstrate a neuroprotective and neurotrophic effect of this calcium-binding protein. The purpose of this study was to examine the role of increased S100B levels on functional outcome after TBI. Following lateral fluid percussion or sham injury in male Sprague Dawley rats (n = 56), we infused S100B (50 ng/h) or vehicle into the cerebrospinal fluid of the ipsilateral ventricle for 7 days using an osmotic mini-pump. Assessment of cognitive performance by the Morris water maze on days 30-34 after injury revealed an improved performance of injured animals after S100B infusion (p < 0.05), when compared to vehicle infusion. Blood samples for analysis of clinical markers of brain damage, S100B and neuron specific enolase, taken at 30 min, 3 h, 4 h, 2 days, or 5 days showed a typical peak 3 h after injury (p < 0.01), and higher serum levels correlated significantly with an impaired cognitive recovery (p < 0.01). The correlation of higher serum S100B levels with poor water maze performance may result from injury induced opening of the blood-brain barrier, allowing the passage of S100B into serum. Thus while higher serum levels of S100B seem to reflect the degree of blood-brain barrier opening and severity of injury, a beneficial effect of intraventricular S100B administration on long-term functional recovery after TBI has been demonstrated for the first time. The exact mechanism by which S100B exerts its neuroprotective or neurotrophic influence remains unknown and needs to be elucidated by further investigation.

Role of aromatic amino acids in carbohydrate binding of plant lectins: Laser photo chemically induced dynamic nuclear polarization study of hevein domain-containing lectins

Carbohydrate recognition by lectins often involves the side chains of tyrosine, tryptophan, and histidine residues. These moieties are able to produce chemically induced dynamic nuclear polarization (CIDNP) signals after laser irradiation in the presence of a suitable radical pair‐generating dye. Elicitation of such a response in proteins implies accessibility of the respective groups to the light‐absorbing dye. In principle, this technique is suitable to monitor surface properties of a receptor and the effect of ligand binding if CIDNP‐reactive amino acids are affected. The application of this method in glycosciences can provide insights into the protein‐carbohydrate interaction process, as illustrated in this initial study. It focuses on a series of N‐acetylglucosamine‐binding plant lectins of increasing structural complexity (hevein, pseudohevein, Urtica dioica agglutinin and wheat germ agglutinin and its domain B), for which structural NMR‐ or X‐ray crystallographic data permit a decision of the validity of the CIDNP method‐derived conclusions. On the other hand, the CIDNP data presented in this study can be used for a rating of our molecular models of hevein, pseudohevein, and domain B obtained by various modeling techniques. Experimentally, the shape and intensity of CIDNP signals are determined in the absence and in the presence of specific glycoligands. When the carbohydrate ligand is bound, CIDNP signals of side chain protons of tyrosine, tryptophan, or histidine residues are altered, for example, they are broadened and of reduced intensity or disappear completely. In the case of UDA, the appearance of a new tryptophan signal upon ligand binding was interpreted as an indication for a conformational change of the corresponding indole ring. Therefore, CIDNP represents a suitable tool to study protein‐carbohydrate interactions in solution, complementing methods such as X‐ray crystallography, high‐resolution multidimensional nuclear magnetic resonance, transferred nuclear Overhauser effect experiments, and molecular modeling. Proteins 28:268–284, 1997

Chronic ethanol consumption transiently reduces adult neural progenitor cell proliferation

Adult neural stem/progenitor cells proliferate throughout the life of the animal in the subependymal zone and the subgranular zone of the dentate gyrus (DG). Treatments such as enriched environment, dietary restriction, running and anti-depressants increase proliferation, however, stress and opiates have been shown to decrease proliferation. While models of binge ethanol drinking decreases proliferation, few studies have characterized the effect chronic ethanol usage has on progenitor cell proliferation. In this study, we have examined changes in the progenitor cell proliferation rate following chronic ethanol consumption. Animals were given a nutritionally balanced liquid diet containing 6.5% v/v ethanol or an isocalorically balanced liquid diet. Bromodeoxyuridine (BrdU) was administered (150 mg/kg x 3) and the animals sacrificed 2 h after the last injection on days 3, 10 or 30 of the ethanol diet. Coronal brain blocks were paraffin embedded and 6 microm sections sliced and immunohistochemically stained for BrdU. Quantitation of the number of BrdU-labeled cells in the subgranular zone of the DG revealed a significant decrease only at the 3-day time-point, with recovery by the 10- and 30-day time-points. Thus, the progenitor cell proliferation rate is transiently decreased by chronic ethanol usage. This data suggests that chronic alcohol use results in a compensatory response that restores the progenitor cell proliferation rate.

Minocycline Blocks Acute Bilirubin-Induced Neurological Dysfunction in Jaundiced Gunn Rats

Background: Extreme hyperbilirubinemia is treated with double volume exchange transfusion, which may take hours to commence. A neuroprotective agent that could be administered immediately might be clinically useful. Minocycline, an anti-inflammatory and anti-apoptotic semisynthetic tetracycline, prevents hyperbilirubinemia-induced cerebellar hypoplasia in Gunn rats. Acute brainstem auditory evoked potential (BAEP) abnormalities occur after giving sulfadimethoxine to 16-day-old jaundiced Gunn rats to displace bilirubin into tissue including brain. Objective: To assess whether minocycline is neuroprotective in this model of acute bilirubin encephalopathy. Methods: We recorded BAEPs at baseline and 6 h after injecting sulfadimethoxine. Minocycline 0.5 mg/kg (n = 4), 5 mg/kg (n = 9), 50 mg/kg (n = 9) or 500 mg/kg (n = 3, all died) was administered 15 min before sulfadimethoxine (0 h). Controls received saline followed by either sulfadimethoxine (n = 13) or saline (n = 7). Results: At 6 h total plasma bilirubin decreased from 10.84 ± 0.88 mg/dl (mean ± SD) to 0.70 ± 0.35 mg/dl (p <10–9) in all sulfadimethoxine-injected groups. At 6 h, there was complete protection against decreased amplitudes of BAEP waves II and III and increased I-II and I-III interwave intervals (brainstem conduction times corresponding to I–III and I–V in humans) with 50 mg/kg minocycline, and partial protection with lower doses. Conclusions: Minocycline 50 mg/kg 15 min prior to an intervention that normally produces acute bilirubin neurotoxicity is neuroprotective in jaundiced Gunn rat pups. Further studies are needed to investigate the temporal course and mechanism of neuroprotection. Minocycline, administered immediately, may be clinically useful in treating extreme neonatal hyperbilirubinemia and preventing kernicterus. We believe our model provides an efficient in vivomodel to screen and evaluate new agents that are neuroprotective against bilirubin toxicity and kernicterus.

Mitochondrial DNA Copy Numbers in Pyramidal Neurons are Decreased and Mitochondrial Biogenesis Transcriptome Signaling is Disrupted in Alzheimer's Disease Hippocampi

Alzheimers disease (AD) is the major cause of adult-onset dementia and is characterized in its pre-diagnostic stage by reduced cerebral cortical glucose metabolism and in later stages by reduced cortical oxygen uptake, implying reduced mitochondrial respiration. Using quantitative PCR we determined the mitochondrial DNA (mtDNA) gene copy numbers from multiple groups of 15 or 20 pyramidal neurons, GFAP(+) astrocytes and dentate granule neurons isolated using laser capture microdissection, and the relative expression of mitochondrial biogenesis (mitobiogenesis) genes in hippocampi from 10 AD and 9 control (CTL) cases. AD pyramidal but not dentate granule neurons had significantly reduced mtDNA copy numbers compared to CTL neurons. Pyramidal neuron mtDNA copy numbers in CTL, but not AD, positively correlated with cDNA levels of multiple mitobiogenesis genes. In CTL, but not in AD, hippocampal cDNA levels of PGC1α were positively correlated with multiple downstream mitobiogenesis factors. Mitochondrial DNA copy numbers in pyramidal neurons did not correlate with hippocampal Aβ1-42 levels. After 48 h exposure of H9 human neural stem cells to the neurotoxic fragment Aβ25-35, mtDNA copy numbers were not significantly altered. In summary, AD postmortem hippocampal pyramidal neurons have reduced mtDNA copy numbers. Mitochondrial biogenesis pathway signaling relationships are disrupted in AD, but are mostly preserved in CTL. Our findings implicate complex alterations of mitochondria-host cell relationships in AD.

A New Animal Model of Hemolytic Hyperbilirubinemia-Induced Bilirubin Encephalopathy (Kernicterus)

Neonatal hyperbilirubinemia can cause bilirubin encephalopathy (kernicterus). Spontaneously jaundiced (jj) Gunn rats treated with sulfonamide (sulfa) to displace bilirubin from serum albumin, develop bilirubin encephalopathy and abnormal brainstem auditory evoked potentials (BAEPs) comparable with human newborns. We hypothesized phenylhydrazine (PHZ)-induced hemolysis would significantly elevate total plasma bilirubin (TB) in jj Gunn rat pups and produce BAEP abnormalities similar to those observed after sulfa. PHZ 0, 25, 50, or 75 mg/kg was administered intraperitonealy to 15-d-old jjs. An initial TB was recorded in each animal, and a second recorded 1–4 d postinjection to generate a dose-response curve. After PHZ 75 mg/kg, TB peaked at about 30 mg/dL at 48–72 h. A second group of jjs injected with PHZ (0, 25, 50, or 75 mg/kg) and nonjaundiced controls given PHZ 75 mg/kg had HCT and TB at baseline, and HCT, TB, and BAEPs recorded at 48 h. BAEP wave II and III amplitudes decreased, and I–II and I–III interwave intervals increased indicating abnormal central (brainstem) auditory function. PHZ-induced hemolysis in jaundiced Gunn rat pups produces sufficiently elevated TB levels to produce bilirubin encephalopathy. This new model may be a more clinically relevant experimental model of kernicterus- and bilirubin-induced neurologic disorders.

Profile of minocycline neuroprotection in bilirubin-induced auditory system dysfunction.

Excessive hyperbilirubinemia in human neonates can cause permanent dysfunction of the auditory system, as assessed with brainstem auditory evoked potentials (BAEPs). Jaundiced Gunn rat pups (jjs) exhibit similar BAEP abnormalities as hyperbilirubinemic neonates. Sulfadimethoxine (sulfa) administration to jjs, which displaces bilirubin from serum albumin into tissues including brain, exacerbates acute toxicity. Minocycline administered prior to sulfa in jjs protects against BAEP abnormalities. This study evaluates the neuroprotective capabilities of minocycline HCl (50 mg/kg) administered 30 or 120 min after sulfa (200 mg/kg) in 16 days old jjs. BAEPs are recorded at 6 or 24 h post-sulfa. Abnormal BAEP waves exhibit increased latency and decreased amplitude. The sulfa/saline treated jjs exhibited a significantly increased interwave interval between waves I and II (I-II IWI) and significantly decreased amplitudes of waves II and III compared to the saline/saline jjs. The minocycline 30 min post-sulfa (sulfa/mino+30) group was not significantly different from the saline/saline control group, indicating neuroprotection. The minocycline 120 min post-sulfa (sulfa/mino+120) group had a significantly decreased amplitude of wave III at both 6 and 24h. These studies indicate that minocycline has a graded neuroprotective effect when administered after acute bilirubin neurotoxicity.