Low-level light in combination with metabolic modulators for effective therapy of injured brain

Vascular damage occurs frequently at the injured brain causing hypoxia and is associated with poor outcomes in the clinics. We found high levels of glycolysis, reduced adenosine triphosphate generation, and increased formation of reactive oxygen species and apoptosis in neurons under hypoxia. Strikingly, these adverse events were reversed significantly by noninvasive exposure of injured brain to low-level light (LLL). Low-level light illumination sustained the mitochondrial membrane potential, constrained cytochrome c leakage in hypoxic cells, and protected them from apoptosis, underscoring a unique property of LLL. The effect of LLL was further bolstered by combination with metabolic substrates such as pyruvate or lactate both in vivo and in vitro. The combinational treatment retained memory and learning activities of injured mice to a normal level, whereas other treatment displayed partial or severe deficiency in these cognitive functions. In accordance with well-protected learning and memory function, the hippocampal region primarily responsible for learning and memory was completely protected by combination treatment, in marked contrast to the severe loss of hippocampal tissue because of secondary damage in control mice. These data clearly suggest that energy metabolic modulators can additively or synergistically enhance the therapeutic effect of LLL in energy-producing insufficient tissue–like injured brain.     

Sequential determinations of cerebrospinal fluid lactate dehydrogenase isoenzyme in human brain tumors on treatment

51 sequential determinations of cerebrospinal fluid (CSF) lactate dehydrogenase (LDH) isoenzyme in 5.5% polyacrylamide gel were performed in 26 samples from patients with brain tumors. The assay was made prior to and after treatment. 26 untreated patients demonstrated elevated total LDH activity. 17 of 26 patients showed altered isoenzyme pattern. Disappearance of LDH₅, decrease of LDH₄ and relative increase of LDH₂ were accompanied by improvement in clinical status in 15 of 26 patients. This suggests the utility of the CSF LDH isoenzyme pattern to monitor the clinical status of treatment in brain tumors.     

Quantitative histochemical changes in enzymes involved in energy metabolism in the rat brain during postnatal Development. I. Cytochrome oxidase and lactate dehydrogenase

The postnatal maturation of cytochrome oxidase and lactate dehydrogenase activity was assessed by histochemistry in rats at 8 postnatal stages, P0, P5, P10, P14, P17, P21, P35 and the adult stage. Enzyme activities were revealed on cryostat brain sections with diaminobenzidine for cytochrome oxidase and nitroblue tetrazolium for lactate dehydrogenase. Lactate dehydrogenase activity remained unchanged between P0 and P10, significantly increased in 8 areas of the 14 studied between P10 and P14 and in 6 structures from P14 to P17. These were mainly parietal, auditory and cerebellar cortices, hippocampus, thalamus, hypothalamus and medial geniculate body. There was no further change until P35 and lactate dehydrogenase activity increased then significantly to reach higher adult levels in hippocampus and medial geniculate body. Cytochrome oxidase activity was low from P0 to P10 and increased in 8 regions between P10 and P14. These were all cortices, caudate nucleus, hippocampus, inferior colliculus and genu. Enzyme activity further increased between P14 and P17 in auditory cortex, medial geniculate body and brainstem, did not vary from P17 to P21 but increased by 92 to 371% in all areas between P21 and P35. Cytochrome oxidase activity rose further from P35 to adult stage in hippocampus and medial geniculate body. From birth to adulthood, cytochrome oxidase activity increased 5 to 19 fold and lactate dehydrogenase activity 1.8 to 3.0. The present study shows that there is a quite good correlation between postnatal changes in regional cerebral glucose utilization and activity of enzymes involved in glycolytic and oxidative glucose metabolism in the rat.     

Transport of proteolipid protein to the plasma membrane does not depend on glycosphingolipid cotransport in oligodendrocyte cultures

The possibility that transport of proteolipid protein (PLP) from its site of synthesis to the plasma membrane is dependent on cotransport with (sulfo)galacto-cerebrosides was investigated in primary cultured oligodendrocytes and Chinese hamster ovary (CHO) cells expressing PLP. Sulfation was inhibited by growing oligodendrocytes in the presence of a competitive inhibitor of this process, sodium chlorate. Under these circumstances, sulfatide synthesis was inhibited by 85%. Nevertheless, PLP was still delivered to the plasma membrane in quantitative amounts. Furthermore, when PLP was expressed in CHO cells, which normally synthesize very low amounts of galactosyl ceramide (GalCer) and no sulfatide, PLP was transported to the plasma membrane. Moreover, in CHO cells coexpressing PLP and ceramide galactosyl transferase, PLP cell surface labeling was unaltered. Noting that it has been demonstrated that proteins destined for the apical surface of epithelial cells colocalize with glycolipid-enriched microdomains, we isolated detergent-insoluble membrane complexes from cultured oligodendrocytes. We found, however, that most of the PLP is present in the detergent-soluble fraction and, furthermore, that PLP could not be chased into or out of the insoluble fraction. Taken together, these data make it very likely that in oligodendrocytes PLP transport takes place irrespective of the presence of glycosphingolipids GalCer and sulfatide. J. Neurosci. Res. 51:371–381, 1998. © 1998 Wiley-Liss, Inc.     

The dark side of self-focus: brain activity during self-focus in low and high brooders

There are two distinct modes of self-focus: analytical self-focus is abstract, general and evaluative whereas experiential self-focus is concrete, specific and non-evaluative. Using functional magnetic resonance imaging (fMRI), we investigated the neural bases of these two modes of self-focus in relation with brooding, the maladaptive form of rumination. Forty-one French-speaking right-handed healthy young adults (10 men, mean age ± s.d.: 21.8 ± 2.3 years) engaged in analytical and experiential self-focus triggered by verbal stimuli during fMRI. Brooding was measured with the 22-item Rumination Response Style scale. Individuals with lower brooding scores showed greater activation of the posterior cingulate cortex/precuneus during analytical than experiential self-focus, whereas individuals with higher brooding scores did not. This is consistent with the hypothesis that brooding is associated with less control over the nature of the self-focus engaged. These findings may help to refine our understanding of how rumination promotes depression through maladaptive self-focus.     

The effects of acute exercise and high lactate levels on 35% CO2 challenge in healthy volunteers

OBJECTIVE: To test the possible antipanic effects of acute exercise in healthy volunteers exposed to an inhalation of 35% CO2 challenge. METHOD: Twenty healthy subjects in a randomized separate group design, performed exercise in a bicycle ergometer reaching >6 mm of blood lactate and a control condition of minimal activity in the same fashion with no lactate elevation. Immediately afterwards an inhalation of a vital capacity using a mixture of 35% CO2/65% O2 through a mask was given on both conditions. RESULTS: Subjects under the exercise condition reported less panic symptoms than controls after a CO2 challenge on the diagnostic statistical manual-IV (DSM-IV) Panic Symptom List but no difference on the Visual Analogue Anxiety Scale. CONCLUSION: Subjects under the exertion condition had lactate levels comparable with those of lactate infusions but an inhibitory rather than accumulative effect was seen when combined with a CO2 challenge.     

Oscillatory brain activity in vegetative and minimally conscious state during a sentence comprehension task

Patients with altered states of consciousness continue to constitute a major challenge in terms of clinical assessment, treatment and daily management. Furthermore, the exploration of brain function in severely brain-damaged patients represents a unique lesional approach to the scientific study of consciousness. Electroencephalography is one means of identifying covert behaviour in the absence of motor activity in these critically ill patients. Here we focus on a language processing task which assesses whether vegetative (n=10) and minimally conscious state patients (n=4) (vs control subjects, n=14) understand semantic information on a sentence level ("The opposite of black is... white/yellow/nice"). Results indicate that only MCS but not VS patients show differential processing of unrelated ("nice") and antonym ("white") words in the form of parietal alpha (10-12Hz) event-related synchronization and desynchronization (ERS/ERD), respectively. Controls show a more typical pattern, characterized by alpha ERD in response to unrelated words and alpha ERS in response to antonyms.     

A systematic regional study of brain salsolinol levels during and immediately following chronic ethanol ingestion in rats

Regional contents of salsolinol and catecholamines in the brain of normal and ethanol-treated rats were studied. Male Sprague Dawley rats were given ethanol solution as sole drinking fluid for 3, 4, 5 or 6 months. Salsolinol determined by gas chromatography mass spectrometry was found to be present in the hypothalamus and the striatum of control rats. The levels of salsolinol in these regions increased significantly by long-term ethanol drinking and rapidly decreased to control levels following its removal. Salsolinol levels in other regions of rat brain were extremely low or negative and unaltered upon chronic ethanol treatment. In ethanol-treated rats the hypothalamic salsolinol, although generally higher than in the striatum, increased along with the ethanol exposure, whereas the striatal salsolinol was constant during those periods of study. Brain dopamine (DA) and norepinephrine contents remained unaltered during and immediately after chronic ethanol treatments. No correlation of salsolinol levels with DA contents or blood ethanol concentrations was observed. The occurrence of salsolinol in selected areas of rat brain with lack of changes in catecholamine level but as a result of an in vivo formation by long-term ethanol drinking was considered to be due to an alteration of acetaldehyde metabolism in the liver and brain.     

Changes in concentration of cerebrospinal fluid components in patients with traumatic brain injury

Brain injury, like other central nervous system pathologies, causes changes in the composition of the cerebrospinal fluid (CSF). In this study, changes in the concentration of small molecules of the CSF, which are in the minimal micromolar concentration, were observed and monitored using high-resolution proton (NMR) spectroscopy. Twenty-two patients with isolated traumatic brain injuries (TBI) and 15 patients making up the control group were recruited for the study. CSF samples were collected by lumbar puncture from the lumbar subarachnoid space in the patients just before commencement of therapy and on the first, third, seventh and fourteenth days of therapy at the ICU. Forty-four signals of the NMR spectra and NO concentration of the CSF samples were analyzed. The analysis shows that the amino acid and organic acid concentrations change during the therapy and mostly are higher than in the control group. Significant differences in concentration of the analyzed CSF components between the TBI patients and the control group have been noted. The rate of the lactate to pyruvate conversion increased because the L/P ratio showed no significant differences between the TBI group and the control group, while the concentrations of both components were significantly higher in the TBI patients than in the control group. Citrulline, arginine and nitric oxide concentrations were the focus of the analysis. Citrulline concentration changes overlapped NO changes from 0 until 3rd day of therapy, while for the remaining days of observation the NO concentration stabilized at the control level, whereas citrulline concentration significantly decreased.     

Catecholamine levels and turnover during aging in brain regions of male C57BL/6J mice

A radioenzymatic assay is described for measuring brain catecholamines (CA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in the same tissue extract. The [³H]-methylated products are differentially extracted and then acetylated by acetic anhydride, followed by thin layer chromatography in non-basic solvents. Routine sensitivity is 3–5 pg per sample. This assay gave CA levels in brain regions which generally agreed with previous reports.CA levels/mg protein and turnover (after injection ofα-methyl-p-tyrosine) were measured in male C57BL/6J mice at various ages between 4 and 30 months, the average lifespan of male C57BL/6J mice. No region showed progressive age changes in levels or turnover, or changes before midlife, 8–12 months. Brain regions with no evidence of age changes include globus pallidus, zona incerta, substantia nigra, cerebellum, and olfactory bulbs. Small changes (10–25%) were detected in only some regions of mice aged ≥ 24 months; few changes were statistically significant. CA levels and turnover decreased in some samplings of striatum and median eminence-arcuate nucleus. In contrast, DA levels increased in anterior pituitary and possibility in medial preoptic region. DOPAC levels (measured only in rostral striatum) decreased by 20% at 28 months. These results diverge from the larger, progressive decreases of DA reported in human striatum during aging.     

Effects of Lamotrigine on brain nitrite and cGMP levels during focal cerebral ischemia in rats

Glutamate receptor antagonists are protective in animal models of focal cerebral ischemia. Lamotrigine (3,5-diamino-6-[2,3-dichlorophenyl]-1,2,4-triazine) is an anticonvulsant drug that blocks voltage-gated sodium channels and inhibits the ischemia-induced release of glutamate. Experiments in primary neuronal cultures implicate nitric oxide (NO) as a mediator of glutamatergic neurotoxicity acting via N-Methyl- d -Aspartate (NMDA) receptors. The effect of glutamate release inhibitor, Lamotrigine upon NO and cGMP production has been examined in focal cerebral ischemia in rats. Focal cerebral ischemia was produced by the permanent occlusion of right middle cerebral artery (MCA) in urethane anesthetized rats. A number of indicators of brain NO production (nitrite, cGMP) were determined in ipsilateral and contralateral cerebral cortex and cerebellum after 0, 10, 60 min of focal cerebral ischemia. The same parameters were measured in rats treated with Lamotrigine (20 mg/kg, i.p.) 30 min before or just after the occlusion of the right MCA.     

Effects of pontine lesions on brain stem polyneuronal activity during sleep in infant rats

Multiple-unit activity was recorded from residual neurons in the gigantocellular field (FTG) of the pontine reticular formation in young rats after extensive contralateral FTG lesions. Epochs of normal appearing active sleep continued to occur but were characterized by abnormally low FTG neuronal firing rates. In contrast, during epochs of active sleep with exaggerated motility, the FTG discharge frequencies approximated those observed during active wakefulness.     

Effects of the triple reuptake inhibitor amitifadine on extracellular levels of monoamines in rat brain regions and on locomotor activity

Major depressive disorder is a prevalent disease, and current pharmacotherapy is considered to be inadequate. It has been hypothesized that a triple reuptake inhibitor (TRI) that activates dopamine (DA) neurotransmission in addition to serotonin and norepinephrine (NE) circuitries may result in enhanced antidepressant effects. Here, we investigated the pharmacological effects of a serotonin-preferring TRI-amitifadine (EB-1010, formerly DOV 21947). The effects of amitifadine (10 mg/kg ip.) on extracellular concentrations of monoamines and their metabolites in rat brain regions were investigated using the in vivo microdialysis technique. The effects of amitifadine on locomotor activity and stereotyped behavior were also evaluated. A major metabolite of amitifadine, the 2-lactam compound, was investigated for inhibition of monoamine uptake processes. Amitifadine markedly and persistently increased extracellular concentrations of serotonin, NE, and DA in prefrontal cortex. The extracellular concentrations of DA were also increased in the DA-rich areas striatum and nucleus accumbens. The extracellular concentrations of the metabolites of serotonin, 5-hydroxyindoleacetic acid, and DA, 3,4-dihydroxyphenylacetic and homovanillic acid, were also markedly decreased in brain regions. Amitifadine did not increase locomotor activity or stereotypical behaviors over a broad dose range. The lactam metabolite of amitifadine weakly inhibited monoamine uptake. Thus, amitifadine increased extracellular concentrations of serotonin, NE, and DA, consistent with TRI. Although amitifadine significantly increased DA in the nucleus accumbens, it did not induce locomotor hyperactivity or stereotypical behaviors. The enhancement of serotonin, NE, and DA in rat brain regions associated with depression suggest that amitifadine may have novel antidepressant activity.     

The effect of duration of cerebral ischemia on brain pyruvate dehydrogenase activity, energy metabolites, and blood flow during reperfusion in gerbil brain

The objective of this study was to determine whether the duration of an ischemic insult effects the activity of the mitochondrial enzyme pyruvate dehydrogenase (PDH) in relation to the recovery of metabolites and regional cerebral blood flow (rCBF) immediately after ischemia and during reperfusion in gerbil cortex. Cerebral ischemia was induced, using the bilateral carotid artery occlusion method, for 20 or 60 min, followed by reperfusion up to 120 min. Immediately after ischemia PDH activity increased threefold regardless of ischemic duration. In the 60-min ischemic group, PDH remained activated, the recovery of high energy phosphates and the clearance of lactate were poor, and the rCBF was 48% of controls after 20-min reperfusion, decreasing gradually to 26% at 120-min reperfusion. In the 20-min ischemic group, PDH activity normalized quickly, the restoration of energy phosphates was good, there was a quick reduction in lactate within the first 60 min of reperfusion, and the rCBF was 65% of control at 20-min reperfusion, and remained over 48% of control throughout reperfusion. Recovery of metabolism after reperfusion did not parallel the changes in rCBF in either group, most noticeably in the 60-min ischemic group. The slow normalization of PDH activity reflected the poor recovery of metabolites in the 60-min ischemic group, indicating that PDH activity is important in the resynthesis of energy metabolites during reperfusion. In conclusion, prolonging the ischemic insult effected PDH activity during reperfusion, impaired recovery of energy metabolites, and worsened the recovery of rCBF.     

Effect of the MAO-B inhibitor,MDL72974, on superoxide dismutase activity and lipid peroxidation levels in the mouse brain

MDL72974 is a member of a series of MAO-B inhibitors to be used as potential therapeutic agents in the treatment of Parkinson's and Alzheimer's diseases. However, we have recently observed a reduction in the density of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra of mice treated with MDL72974. As oxidative stress is known to play a significant role in the nigrostriatal pathway, analysis of the relationship between TH⁺ cell losses induced by MDL72974 and by free radical production was investigated in the present study. Results demonstrate a significant increase in superoxide dismutase (SOD) activity, a key antioxidant, in the striatum and cerebellum of MDL72974-treated mice, presumably in response to free radical production. An increase in lipid peroxidation levels was also observed in the striatum of these animals in a manner which is consistent with oxidative stress-inducing agents. We therefore suggest that MDL72974 may be detrimental to dopaminergic neurons of the nigrostriatal pathway via free radical-mediated reactions. Synapse 28:208–211, 1998. © 1998 Wiley-Liss, Inc.     

The influence of hypothalamic proline-rich polypeptide on ATP-phosphohydrolase activity in rat brain mitochondria during epileptiform seizures induced by pentylenetetrazole

We have studied the shifts in the activity of Mg²⁺-, Ca²⁺-, and HCO ₃ ^? -dependent ATPases in brain mitochondria of white rats during epileptiform seizures (ES) induced by pentylenetetrazole. Inhibition of the catalytic activity of Mg²⁺-, Ca²⁺-, and HCO ₃ ^? -dependent ATPases was observed during ES. However, a preliminary intraperitoneal injection of proline-rich polypeptide (PRP) restored the activity of the investigated enzymes after ES to the control level. It is possible to assume that PRP has a protective effect on the body intoxication caused by pentylenetetrazole.     

Photic inhibition of TrkB/Ras activity in the pigeon's tectum during development: impact on brain asymmetry formation

Asymmetric photic stimulation during embryonic or post-hatch development induces a functional lateralization of the pigeon's visual system, which is accompanied by left-right differences in tectal cell sizes. The intracellular membrane-anchored GTPase Ras can be activated by a number of upstream mechanisms including binding of brain-derived neurotrophic factor to its specific TrkB receptor. Ras activity plays an important morphogenetic role in neurons and therefore might also be involved in the asymmetric differentiation of tectal cells. To investigate the role of Ras, we determined the relative levels of activated Ras and of signalling active phospho-TrkB in tecta of light- and dark-incubated pigeons and combined this with an immunohistochemical detection of Ras-GTP and TrkB receptors. While Ras activation levels did not differ between light- and dark-incubated pigeons during embryonic development, directly after hatching Ras activity was significantly decreased in the stronger stimulated left tectum of light-incubated animals. This was accompanied by lower levels of TrkB phosphorylation. Immunohistochemical staining revealed Ras-GTP-positive cell bodies within the efferent cell layer. These cells were TrkB-positive and developed enlarged soma sizes within the right tectum during the first week after hatching. This association suggests asymmetric Ras activation to be involved in the asymmetric differentiation of the efferent cells as a result of asymmetric TrkB signalling. Because asymmetric light exposure occurs only during embryonic development, the observed transient asymmetric inhibition of TrkB/Ras activity after hatching may reflect differential embryonic maturation of tectal inhibitory circuits leading to a functional superiority of the right eye in the adult organism.     

Functional brain plasticity during L1 training on complex sentences: Changes in gamma‐band oscillatory activity

The adult human brain remains plastic even after puberty. However, whether first language (L1) training in adults can alter the language network is yet largely unknown. Thus, we conducted a longitudinal training experiment on syntactically complex German sentence comprehension. Sentence complexity was varied by the depth of the center embedded relative clauses (i.e., single or double embedded). Comprehension was tested after each sentence with a question on the thematic role assignment. Thirty adult, native German speakers were recruited for 4 days of training. Magnetoencephalography (MEG) data were recorded and subjected to spectral power analysis covering the classical frequency bands (i.e., theta, alpha, beta, low gamma, and gamma). Normalized spectral power, time‐locked to the final closure of the relative clause, was subjected to a two‐factor analysis (“sentence complexity” and “training days”). Results showed that for the more complex sentences, the interaction of sentence complexity and training days was observed in Brodmann area 44 (BA 44) as a decrease of gamma power with training. Moreover, in the gamma band (55–95 Hz) functional connectivity between BA 44 and other brain regions such as the inferior frontal sulcus and the inferior parietal cortex were correlated with behavioral performance increase due to training. These results show that even for native speakers, complex L1 sentence training improves language performance and alters neural activities of the left hemispheric language network. Training strengthens the use of the dorsal processing stream with working‐memory‐related brain regions for syntactically complex sentences, thereby demonstrating the brain''s functional plasticity for L1 training.