Trace fear conditioning detects hypoxic-ischemic brain injury in neonatal mice

Trace fear conditioning is a well-established test for the assessment of learning deficits in rodents. The aim of this study was to determine whether hypoxia-ischemia (HI) on postnatal day 9 (P9) in mice prevents the acquisition and expression of cued and contextual fear learning in early adulthood. Brain injury was induced in mice on P9 by 30 min of HI. On P49 and P50, animals were tested for: (1) trace fear conditioning with a short delay (2 s) between a shock-paired tone plus light and shock, (2) trace fear conditioning with a longer delay (20 s) between a shock-paired tone and shock, and (3) trace fear conditioning with a 2-second delay between a shock-paired tone and shock with additional visual, olfactory and tactile contextual cues in the fear conditioning apparatus. Outcome was assessed as percent of time spent freezing during a 2-min test. Histological assessment of the hippocampus and amygdala was performed on P51 to determine the extent of HI injury. Both shock-paired tone plus light with a short delay and shock-paired tone with a short delay plus additional contextual cues enhanced tone-induced freezing behavior in a nonhandled control group, but not in the HI group. For trace fear conditioning with a 20-second delay between the tone and the shock, freezing behavior did not differ significantly between nonhandled control and HI animals. Dorsal hippocampal and amygdala volumes were smaller in the ischemic hemispheres of the HI mice that displayed impaired fear memory with shock-paired tone plus light. In summary, we have shown that trace fear conditioning is a sensitive method for detecting memory impairments in adolescent mice following mild HI injury during the neonatal period. Combining a discrete conditioned stimulus (shock-paired tone plus light) with a short trace delay was the most sensitive method for using the fear conditioning paradigm to detect mild HI damage to the hippocampus and amygdala.     

Optical imaging of brain function and metabolism

Summary Optical techniques already have an important place in experimental in vivo studies of the brain. New techniques to study micro circulatory and metabolic events in vivo will play an important role in basic stroke research. Functional studies are rapidly improving our knowledge of the functional architecture of the visual cortex. For studies of the human brain the confluence of interest and activity in MRI, PET, ballistic and diffusive wave spectroscopy opens new fields of anatomical, physiological, biochemical and functional studies. Similar to the introduction of the morphologically oriented computed tomography and magnetic resonance imaging, these new approaches may have a profound impact on clinical and experimental neurology in the future.The proceedings of this symposium will be published within the seriesAdvances in Experimental Medicine and Biology, Plenum Press, London New York, fall 1992.     

EAE大鼠外周免疫器官细胞凋亡的研究

目的:研究实验性变态反应性脑脊髓炎(EAE)大鼠外周免疫器官中细胞凋亡的情况。方法:应用脱氧核糖核苷酸末端转移酶介导的缺口末端标记(TUNEL)技术和流式细胞仪定量分析动态检测EAE病程不同阶段大鼠外周免疫器官中细胞凋亡的变化情况。结果:实验组脾和淋巴结中细胞凋亡率很低,组间及组内均无显著差异。结论:外周免疫器官在EAE凋亡中不起主要作用。     

Distribution of pipecolic acid and proline in the developing rat brain and peripheral organs

The regional distribution of pipecolic acid and proline was studied in the adult and newborn rat brain. The brain concentration of pipecolic acid in the 1 and 3-day-old rat was 6–10 times higher than in the adult. The brain concentration of proline was found to be about 3 times higher in the brain of a newborn rat than in the adult, then it gradually decreased during the period of lactation. In plasma as well as in peripheral tissues, such as heart, kidney and liver, the highest concentration of pipecolic acid was observed at 1 day and in the adult. Secretion of pipecolic acid into the urine was highest at early stages of development. In the gastrointestinal tract, pipecolic acid was found to be most abundant in the adult, whereas low levels were found during the period of lactation. The developmental and regional changes in concentration of proline were quite different from those of pipecolic acid. Our results suggest that the contribution of dietary pipecolic acid in building up levels of pipecolic acid in rat brain is small.     

Ischemic preconditioning increases antioxidants in the brain and peripheral organs after cerebral ischemia

BACKGROUND AND PURPOSE: Low molecular weight antioxidants (LMWA), which reflect tissue reducing power, are among the endogenous mechanisms for neutralizing reactive oxygen species (ROS). Ischemic preconditioning (IPC) was associated with decreased oxidative stress. We examined the effect of focal ischemia on LMWA and on prostaglandin E(2) (PGE(2), a product of arachidonic acid oxidation) in the brain, heart, liver, and lungs of rats subjected to 90 min of ischemia and in IPC rats subjected to similar insult. METHODS: Transient right middle cerebral artery occlusion (MCAO) was performed for 90 min and at 0, 5, 30, 60, or 240 min of reperfusion, LMWA and PGE(2) were evaluated by cyclic voltametry (CV) and radioimmunoassay, respectively. IPC was induced by 2 min of MCAO, 24 h prior to the major ischemic episode. RESULTS: LMWA decreased at 5 min of reperfusion in the brain, heart, liver, and lung and rose 4 h later only in the brain. PGE(2) levels increased three to fivefold in all tissues examined. Surprisingly, in IPC rats a dramatic increase of LMWA occurred at 5 min of reperfusion in the brain and in the peripheral organs. Uric acid, but not ascorbic, is the major LMWA increased. CONCLUSIONS: We propose that after ischemia, ROS rapidly consume the antioxidants reserves in the brain and also in peripheral organs, suggesting that the whole body is under oxidative stress. Moreover, part of the neuroprotection afforded by IPC is mediated by the brain's ability to mobilize antioxidants, especially uric acid, that attenuate the massive ROS-mediated oxidative stress.     

Monoamine oxidase A imaging in peripheral organs in healthy human subjects

Monoamine oxidase (MAO) catalyzes the oxidative deamination of many biogenic and dietary amines. Though studies of MAO have focused mainly on its regulatory role in the brain, MAO in peripheral organs also represents a vast mechanism for detoxifying vasoactive compounds as well as for terminating the action of physiologically active amines, which can cross the blood brain barrier. Indeed, robust central and peripheral MAO activity is a major requirement in the safe use of many CNS drugs, particularly antidepressants, and thus an awareness of the MAO inhibitory potential of drugs is essential in therapeutics. In this study, we examined the feasibility of quantifying MAO A in peripheral organs in healthy human subjects using comparative positron emission tomography (PET) imaging with carbon-11 (t(1/2): 20.4 min) labeled clorgyline ([(11)C]clorgyline) a suicide inactivator of MAO A and its deuterium labeled counterpart ([(11)C]clorgyline-D2). Heart, lungs, kidneys, thyroid, and spleen showed a robust deuterium isotope effect characteristic of MAO and the magnitude of the effect was similar to that of trancylcypromine, an irreversible MAO inhibitor used in the treatment of depression. Liver time-activity curves were not affected by deuterium substitution precluding the estimation of liver MAO in vivo. In organs showing an isotope effect, MAO A is greatest in the lungs and kidneys followed by the thyroid and heart. This method, which has been previously applied in the human brain, opens the possibility to also directly assess the effects of different variables including smoking, dietary substances, drugs, disease, and genetics on peripheral MAO A in humans.     

Decreased MK-801 binding in discrete hippocampal regions of prion-infected mice

The neurochemical alterations associated with neurodegeneration in prion diseases are not well defined. It is therefore of interest to study the influence of prion infection on messenger molecules and their receptors. In the present study we have analyzed the possible involvement of NMDA receptors in prion-infected mice using ligand binding autoradiography and iodinated MK-801, a noncompetitive NMDA antagonist. The results show a reduced binding of MK-801 in discrete regions of hippocampus at 110 days after infection, that is before the appearance of behavioral symptoms. In addition, early transient increases in MK-801 binding were observed in several layers. The exact neuroanatomical correlate of these changes in MK-801 binding, as well as its functional significance in relation to prion symptomatology, remain to be analyzed.     

Differential distribution of cAMP-specific phosphodiesterase 7A mRNA in rat brain and peripheral organs

We investigated the regional distribution and cellular localization of mRNA coding for the cAMP-specific phosphodiesterase 7A (PDE7A) in rat brain and several peripheral organs by in situ hybridization histochemistry. The regional expression of two splice variants, PDE7A1 and PDE7A2, was examined by RT-PCR using RNA extracted from several brain regions. PDE7A mRNA was found to be widely distributed in rat brain in both neuronal and nonneuronal cell populations. The highest levels of hybridization were observed in the olfactory bulb, olfactory tubercle, hippocampus, cerebellum, medial habenula nucleus, pineal gland, area postrema, and choroid plexus. Positive hybridization signals were also detected in other areas, such as raphe nuclei, temporal and entorhinal cortex, pontine nuclei, and some cranial nerve motor nuclei. Both mRNA splice forms were differentially distributed in several areas of the brain with the striatum expressing only PDE7A1 and the olfactory bulb and spinal cord expressing PDE7A2 exclusively. In peripheral organs the highest levels of PDE7A hybridization were seen in kidney medulla, although testis, liver, adrenal glands, thymus, and spleen also presented high hybridization signal. These results are consistent with PDE7A being involved in the regulation of cAMP signaling in many brain functions. The consistent colocalization with PDE4 mRNAs suggests that PDE7A could have an effect on memory, depression, and emesis. The results offer clear anatomical and functional systems in which to investigate future specific PDE7 inhibitors.     

Increased uptake of the P2X7 receptor radiotracer 18F-JNJ-64413739 in the brain and peripheral organs according to the severity of status epilepticus in male mice

Objective

The P2X7 receptor (P2X7R) is an important contributor to neuroinflammation, responding to extracellularly released adenosine triphosphate. Expression of the P2X7R is increased in the brain in experimental and human epilepsy, and genetic or pharmacologic targeting of the receptor can reduce seizure frequency and severity in preclinical models. Experimentally induced seizures also increase levels of the P2X7R in blood. Here, we tested ¹⁸F-JNJ-64413739, a positron emission tomography (PET) P2X7R antagonist, as a potential noninvasive biomarker of seizure-damage and epileptogenesis.

Methods

Status epilepticus was induced via an intra-amygdala microinjection of kainic acid. Static PET studies (30 min duration, initiated 30 min after tracer administration) were conducted 48 h after status epilepticus via an intravenous injection of ¹⁸F-JNJ-64413739. PET images were coregistered with a brain magnetic resonance imaging atlas, tracer uptake was determined in the different brain regions and peripheral organs, and values were correlated to seizure severity during status epilepticus. ¹⁸F-JNJ-64413739 was also applied to ex vivo human brain slices obtained following surgical resection for intractable temporal lobe epilepsy.

Results

P2X7R radiotracer uptake correlated strongly with seizure severity during status epilepticus in brain structures including the cerebellum and ipsi- and contralateral cortex, hippocampus, striatum, and thalamus. In addition, a correlation between radiotracer uptake and seizure severity was also evident in peripheral organs such as the heart and the liver. Finally, P2X7R radiotracer uptake was found elevated in brain sections from patients with temporal lobe epilepsy when compared to control.

Significance

Taken together, our data suggest that P2X7R-based PET imaging may help to identify seizure-induced neuropathology and temporal lobe epilepsy patients with increased P2X7R levels possibly benefitting from P2X7R-based treatments.     

Evaluation of a new norepinephrine transporter PET ligand in baboons, both in brain and peripheral organs

Reboxetine is a specific norepinephrine transporter (NET) inhibitor and has been marketed in several countries as a racemic mixture of the (R,R) and (S,S) enantiomers for the treatment of depression. Its methyl analog (methylreboxetine, MRB) has been shown to be more potent than reboxetine itself. We developed a nine-step synthetic procedure to prepare the normethyl precursor, which was used to synthesize [11C]O-methylreboxetine ([11C]MRB). We also developed a convenient resolution method using a chiral HPLC column to resolve the racemic precursor to obtain enantiomerically pure individual precursors that lead to the individual enantiomers (R,R)-[11C]MRB and (S,S)-[11C]MRB. Here we report an evaluation of the racemate and individual enantiomers of [11C]MRB as radioligands for PET imaging studies of NET systems in baboons both in brain and in peripheral organs. The relative regional distribution of the radioactivity after injection of [11C]MRB in baboon brain is consistent with the known distribution of NET. For a NET-poor region such as striatum, there were no significant changes in the striatal uptakes with and without the nisoxetine pretreatment. In contrast, a significant blocking effect was observed in NET-rich regions such as thalamus and cerebellum after injection of racemic [11C]MRB, with an even more dramatic effect after injection of (S,S)-[11C]MRB. These results, along with the fact that there was no regional specificity and no blocking effect by nisoxetine for (R,R)-[11C]MRB, suggest the enantioselectivity of MRB in vivo, consistent with previous in vitro and in vivo studies in rodents. PET studies of baboon torso revealed a blocking effect by desipramine only in the heart, a NET-rich organ, after injection of (S,S)-[11C]MRB, but not the (R,R)-isomer. These studies demonstrate that the use of (S,S)-[11C]MRB would allow a better understanding of the role that NET plays in living systems.     

The effects of 6-hydroxydopamine pretreatment on the accumulation of dopa and dopamine in brain and peripheral organs following l-dopa administration

Summary The capacity of brain and peripheral organs to accumulate dopamine following intraperitoneal l-dopa was not impaired in rats whose catecholamine-containing nerve terminals had been destroyed by pretreatment with intracisternal or intraperitoneal 6-hydroxydopamine. These findings indicate that the uptake of exogenous l-dopa and its conversion to dopamine are not restricted to cells that normally synthesize and contain catecholamines.     

Blood borne hormones in a cross-talk between peripheral and brain mechanisms regulating blood pressure,the role of circumventricular organs

Accumulating evidence suggests that blood borne hormones modulate brain mechanisms regulating blood pressure. This appears to be mediated by the circumventricular organs which are located in the walls of the brain ventricular system and lack the blood–brain barrier. Recent evidence shows that neurons of the circumventricular organs express receptors for the majority of cardiovascular hormones. Intracerebroventricular infusions of hormones and their antagonists is one approach to evaluate the influence of blood borne hormones on the neural mechanisms regulating arterial blood pressure. Interestingly, there is no clear correlation between peripheral and central effects of cardiovascular hormones. For example, angiotensin II increases blood pressure acting peripherally and centrally, whereas peripherally acting pressor catecholamines decrease blood pressure when infused intracerebroventricularly. The physiological role of such dual hemodynamic responses has not yet been clarified.     

Optical coherence tomography of the optic nerve head detects acute changes in intracranial pressure

We aimed to determine if there are measurable objective changes in the optic nerve head (ONH) immediately after cerebrospinal fluid (CSF) drainage in a prospective case-series of five patients undergoing a clinically indicated lumbar puncture (LP) for diagnosis of idiopathic intracranial hypertension. A Cirrus high-definition optical coherence tomography machine (Carl Zeiss Meditec, Dublin, CA, USA) was used to acquire images in the lateral decubitus position. Optic disc cube and high-definition line raster scans centered on the ONH were obtained immediately before and after draining CSF, while the patient maintained the lateral decubitus position. Measured parameters included retinal nerve fiber layer (RNFL) thickness, peripapillary retinal pigment epithelium/Bruch’s membrane (RPE/BM) angulation, transverse neural canal diameter (NCD) and the highest vertical point of the internal limiting membrane from the transverse diameter (papillary height). The mean (±standard deviation) opening and closing CSF pressures were 34.3 ± 11.8 and 11.6 ± 3.3 cmH₂O, respectively. Mean RNFL thickness (pre LP: 196 ± 105 μm; post LP: 164 ± 77 μm, p = 0.1) and transverse NCD (pre LP: 1985 ± 559 μm; post LP: 1590 ± 228 μm, p = 2.0) decreased in all subjects, but with non-significant trends. The RPE/BM angle (mean change: 5.8 ± 2.0 degrees, p = 0.003) decreased in all subjects. A decrease in papillary height was seen in three of five subjects (mean: pre LP: 976 ± 275 μm; post LP: 938 ± 300 μm, p = 0.9). Our results show a measurable, objective change in the ONH after acute lowering of the lumbar CSF pressure, suggesting a direct link between the lumbar subarachnoid space and ONH regions, and its potential as a non-invasive method for monitoring intracranial pressures.     

Fibronectin staining detects micro-organisms in aged and Alzheimer's disease brain.

Filamentous, fibronectin-immunopositive structures, previously described in Alzheimer's disease and control brains were negative for neuronal, glial, and macrophage markers. The present study sought to determine the nature of these entities and to further characterize their morphology, immunoreactivity and distribution between neuropathologies. Ultrastructural analysis shows these formations to be filamentous micro-organisms, which may belong to the actinomycetes. Immunohistochemistry for the cell-stress protein ubiquitin is consistently positive in these organisms. They are also present in Down's syndrome, dementia pugilistica, amyotrophic lateral sclerosis with dementia, and Parkinson's disease. The pattern of tissue distribution implies a pre-mortem invasion of the brain, and, as the micro-organism is present at a four to five-fold higher frequency in Alzheimer's disease, it may act pathogenically in this dementing illness.     

Expression of neutral glycosphingolipids in the brain, lymphoid organs and lungs of mice lacking beta(2)-microglobulin

OBJECTIVES AND METHODS:Previous studies have demonstrated the involvement of glycosphingolipid (GSL) antigens in the pathogenesis of immune-mediated neurological disorders such as peripheral neuropathies and multiple sclerosis, as well as in infections of the central nervous system. We investigated the expression of neutral GSLs in the brain, thymus, spleen, and lungs of mice lacking the gene for the beta(2)-microglobulin (beta(2)M), an important component of the MHC class I molecule. Determination of GSL fractions in the tissues of mice homozygous (beta(2)M-/-) or heterozygous (beta(2)M+/-) for beta(2)M gene knockout was performed by high performance thin layer chromatography, followed by immunostaining with specific antibodies. RESULTS: Immunochemical analyses revealed abundant expression of globotetraosylceramide (Gb 4), in the brain of beta(2)M-/- mice, compared with undetectable expression in the control beta(2)M+/- mice. The brain of both groups of animals was negative for Gg 3, Gg 4 and Gb 3 immunostaining. Abundant expression of Gg 3, Gg 4, and Gb 3 was found in the lungs of control beta(2)M+/- mice, whereas beta(2)M-/- mice lacked these structures. Immunostaining of Gb 4 in the lungs was similar in both groups of animals. The thymus and spleen neutral GSL profiles did not significantly differ between beta(2)M-/- and control animals. CONCLUSION: This study provides in vivo-evidence that globo- and ganglio-series neutral GSL expression in the brain and lungs may be related to the presence of beta(2)M or functional MHC class I molecule, and implicates different modulation of biosynthesis of globo- and ganglio-series neutral GSLs in these tissues.     

MRI mean diffusivity detects widespread brain degeneration in multiple sclerosis

We investigated the magnetic resonance imaging (MRI) findings of 32 multiple sclerosis (MS) patients using voxel-based morphometry (VBM) and voxel-based analysis of white matter fluid-attenuated inversion recovery image (FLAIR) high-intensity lesions and diffusion tensor imaging (DTI). Compared with 18 healthy controls, MS patients showed gray matter volume reduction in the thalamus, hypothalamus, caudate, limbic lobe, and frontal lobe. A marked volume reduction of white matter was evident along the ventriculus lateralis and corpus callosum. FLAIR high-intensity lesions were observed beside the ventriculus lateralis. DTI revealed reduced fractional anisotropy areas similar to those of the FLAIR high-intensity lesions. Changes in the volume of increased mean diffusivity (MD) were the most widespread and extended to normal-appearing white matter (p<0.001). Multiple regression analysis revealed that MD values were significantly correlated with both disease duration (r=0.381, p=0.032) and expanded disability status scale scores (EDSS) (r=0.393, p=0.026). This study demonstrated that combined voxel-based analysis for volumetry, FLAIR high-intensity lesions, and DTI could reveal widespread brain abnormalities in MS patients. Furthermore, DTI, especially MD, showed far more widespread brain degeneration than other MRI parameters, and was significantly correlated with both severity and disease duration.