Decrease in N-acetylaspartate/creatine ratio in the motor area and the frontal lobe in amyotrophic lateral sclerosis.

We studied whether N-acetylaspartate (NAA), a neuronal marker, is reduced in the brain of 14 patients with clinically definite amyotrophic lateral sclerosis (ALS) and whether NAA levels in the motor area and frontal lobe correlate with the clinical features, including frontal lobe function. We also studied 14 normal controls were evaluated. We obtained peak integrals in 1H magnetic resonance spectroscopy (MRS) for NAA, creatine (Cr), and choline-containing compounds (Cho). Severity of the disease was determined using the manual muscle strength test, and the Norris limb and bulbar scales. In the patients, the NAA/Cr ratio was reduced in the motor area and frontal lobe, while the Cho/Cr ratio was normal throughout the brain. There were significant correlations between the NAA/Cr ratio in the motor area and the Norris limb scale (r = 0.50; P < 0.01) and between the NAA/Cr ratio in the frontal lobe and the number of categories achieved in the Wisconsin Card Sorting test (r = 0.71; P < 0.05), implying frontal lobe dysfunction. These correlations suggest that a reduced NAA/Cr ratio is a marker of cortical neuronal loss and dysfunction in ALS.

Proton magnetic resonance spectroscopy of patients with parkinsonism.

We studied cerebral metabolism in 82 patients with nonfamilial parkinsonism, including Parkinson's disease (PD; n = 23), progressive supranuclear palsy (PSP; n = 12), corticobasal degeneration (CBD; n = 19), multiple systemic atrophy (MSA; n = 18) and vascular parkinsonism (VP; n = 10) by using proton magnetic resonance spectroscopy ((1)H-MRS), which allowed noninvasive measurement of signal intensities from N-acetylasparate (NAA), choline-containing compounds (CHO) and creatine plus phosphocreatine (CRE). As compared to normal controls, patients with PSP, CBD, MSA and VP, but not PD, had significant reduction of the NAA/CRE ratio in the frontal cortex, whereas patients with PSP, CBD, MSA and PD, but not VP, had significant reduction of the NAA/CRE ratio in the putamen. Patients with CBD had significant reduction of the NAA/CRE ratio in the frontal cortex and putamen as compared to patients with PD, MSA and VP. Patients with PSP showed a significant reduction of the NAA/CRE ratio in the putamen as compared with patients with PD and MSA. Patients with CBD showed clear asymmetry in the putamen as compared to controls and other patients. The reduction of the NAA/CRE ratio in the putamen correlated well with the severity of parkinsonism. (1)H-MRS may be useful in monitoring patients with various types of parkinsonism.

Proton magnetic resonance spectroscopy (1H MRS) in patients with sporadic cerebellar degeneration.

The authors studied 23 patients with cerebellar degeneration including multiple systemic atrophy (MSA) and cerebellar cortical atrophy (CCA) by proton magnetic resonance spectroscopy (1H-MRS). 1H-MRS allowed noninvasive measurement of the signal intensities derived from N-acetylaspartate (NAA), creatine + phosphocreatine (CRE), and choline-containing compounds (CHO). There was significant reduction of the NAA/CRE level in the frontal cortex, putamen, cerebellar hemisphere and cerebellar vermis of patients with MSA, and in the frontal cortex, cerebellar hemisphere and cerebellar vermis of patients with CCA as compared with those of normal controls. There was significant reduction of the NAA/CRE level also in the putamen of patients with MSA as compared with that of patients with CCA. These results indicated the presence of a degenerative process and/or functional impairment in the frontal cortex and putamen of patients with MSA and in the frontal cortex of patients with CCA, in addition to a degenerative process in the cerebellum. There was a significant correlation between the NAA/CRE level and the severity of clinical signs. 1H-MRS is valuable in providing information regarding the pathophysiology and the progress of cerebellar degenerative diseases.

Effect of coenzyme Q10 in patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS): evaluation by noninvasive tissue oximetry.

We evaluated the effect of coenzyme Q10 supplementation to two patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) by using noninvasive tissue oximetry with near-infrared spectra of hemoglobin from the quadriceps muscle during bicycle ergometer exercise. Patients showed distinct oxygen consumption patterns reflecting the defect in oxidative phosphorylation and the impairment in oxygen utilization during exercise. Based on the oxygen consumption pattern, we considered one patient as having severe mitochondrial disorder and another patient as having mild one. After coenzyme Q10 supplementation, the oxygen consumption pattern of the patient with the severe form shifted to the mild one, while that of the patient with mild form remained unchanged. The shift of the pattern to the mild form correlated well with reduction of the sum of the serum lactate and pyruvate content during exercise. Noninvasive tissue oximetry may be useful to evaluate the effect of coenzyme Q10 supplementation to patients with mitochondrial encephalomyopathy including MELAS.

Effect of systemic zinc administration on delayed neuronal death in the gerbil hippocampus.

The divalent cation zinc has been reported to possess several physiological properties such as blocking apoptotic cell death through an inhibitory effect on Ca(2+)-Mg2+ endonuclease activity, or modulating the neurotoxicity via glutamate receptor subtypes. In the present study, we investigated the effect of peripherally injected zinc on delayed neuronal death seen in the hippocampus after transient global ischemia, in order to elucidate a possible beneficial role on zinc in ischemic neuronal cell death. Forty-five adult Mongolian gerbils of both sexes underwent transient bilateral clipping of the common carotid arteries for 3 min. In the pretreated animals, ZnCl2 (20 mg/kg) was injected subcutaneously once, 1 h before ischemia (superacute group; n = 6) or twice at 24 and 48 h before ischemia (subacute group; n = 14). Histological survey was carried out 3 days later by in situ DNA fragmentation method and 4 days later by hematoxylin-eosin staining by semiquantatively counting dead neurons in the CA1 sector. Subacute zinc pre-administration significantly reduced the nuclear damage and subsequent neuronal death; however, superacutely pre-administered zinc did not protect hippocampal neurons against ischemia but it did not aggravate the effect of ischemia, either. The present study suggested that transfer of exogenous zinc into the intracellular space is required for neuroprotection, presumably via the anti-endonuclease activity.

Hemiparkinsonism following haemorrhage in the contralateral substantia nigra.

We report a patient with right hemiparkinsonism following haemorrhage in the left substantia nigra. The hemiparkinsonism responded to treatment with trihexyphenidyl hydrochloride and deteriorated after temporary discontinuation of the drug. Single photon emission computed tomography using technetium 99m d, l-hexamethylpropyleneamine oxide showed reduced uptake in the left putamen, globus pallidus and thalamus.

Induction of c-fos and c-jun gene products and heat shock protein after brief and prolonged cerebral ischemia in gerbils.

BACKGROUND AND PURPOSE: Proto-oncogene activation and induction of heat shock protein (HSP) occur in response to various stimuli to brain, but the role in neuronal survival after cerebral ischemia remains uncertain. We compared the extent of insults and induction of c-fos and c-jun gene products (c-FOS and c-JUN) as well as HSP in ischemic and postischemic gerbil brains immunohistochemically. METHODS: Common carotid arteries of Mongolian gerbils were occluded for 5 or 15 minutes and recirculated for 0 minutes to 7 days. Antibodies for c-FOS, c-JUN, and HSP 70 were used for immunohistochemistry, and positive reactions were semiquantitatively analyzed. The presence of ischemic and postischemic lesions was ascertained with an antibody for microtubule-associated proteins. RESULTS: After ischemia for 15 minutes and reperfusion, c-FOS was induced promptly after 1 to 6 hours in pyramidal cells of the CA3 and CA4 regions, while c-JUN became visible in the same areas after recirculation for 4 to 48 hours. HSP 70 was detected after recirculation for 24 hours in the CA3 region. In layers I and II of the cerebral cortex, c-FOS and c-JUN peaked at 3 hours and HSP 70 at 96 hours. Induction of these proteins was absent or negligible in the areas that developed ischemic or postischemic lesions, including the subiculum-CA1 and CA1 regions of the hippocampus and layers III/IV and Vb/VI of the cerebral cortex. After shorter ischemia for 5 minutes and reperfusion, c-FOS and c-JUN were rapidly induced at 15 minutes to 1 hour except for the subiculum-CA1 and CA1 regions of the hippocampus. Induction of HSP 70 did not occur for 24 hours and was noted only in the hippocampus. CONCLUSIONS: Induction of c-FOS and c-JUN occurred in the areas surviving after transient cerebral ischemia, but the extent of induction and the latent period varied depending on the duration of the insult and the location. In the areas with ischemic or postischemic damage detected by loss of the reaction for microtubule-associated proteins, the induction of c-FOS and c-JUN was either absent or minimal, suggesting that active induction of those immediate early gene products occurred early in surviving neurons. On the other hand, the induction of HSP 70 did not occur until reperfusion for 24 hours and actively occurred only in the areas with earlier induction of c-FOS and/or c-JUN, suggesting that the induction of HSP 70 occurred in neurons that survived to that point, but it did not participate in early responses for neuronal survival after global cerebral ischemia.

Fluoride distribution in dentine and cementum in human permanent teeth with vital and non-vital pulps.

Twenty-two selected permanent teeth [11 with vital pulps and 11 with non-vital (necrotic) pulps] were obtained from patients aged from 20 to 73 yr in Nagoya, Japan. Fluoride (F) profiles in tooth samples were examined by using an abrasive micro-sampling technique. F levels in the pulpal dentine were significantly higher in vital than non-vital teeth for each age group, although there were observed increases of F with age. In the cementum the F levels were strongly related to age and there were few differences in profiles and levels of F between vital and non-vital teeth. It was concluded that vital pulp is an important factor for the pulpal dentine to absorb fluoride.

Mercury and selenium contents in amyotrophic lateral sclerosis in Hokkaido, the northernmost island of Japan.

We evaluated the pathogenicity of mercury (Hg) and selenium (Se) which are supposed to be one of the risk factors in the development of amyotrophic lateral sclerosis (ALS). Hg and Se contents were measured in plasma, blood cells, scalp hair samples of 21 sporadic ALS patients and 36 controls, who included 19 patients with other neurological diseases, in Hokkaido, the northernmost island of Japan. Hg and Se levels in plasma and blood cells of ALS patients were significantly lower in advanced staged ALS patients than controls. Low Hg and Se contents in ALS, being correlated with their disabilities and nutritional conditions, would rather reflect the disease contracted states than the pathogenic roles in ALS.

Immunohistochemical investigation of ischemic and postischemic damage after bilateral carotid occlusion in gerbils.

We investigated progression and recovery of neuronal damage during and after global cerebral ischemia in gerbils after bilateral occlusion of the common carotid arteries, using the immunohistochemical method (reaction for tubulin and creatine kinase BB-isoenzyme). The earliest, but reversible, ischemic lesions occurred after 3 minutes' ischemia in the subiculum-CA1 and CA2 regions of the hippocampus. The lesions became irreversible after 4 minutes' ischemia. The ischemic and postischemic lesions in the cerebral cortex, thalamus, and caudoputamen were partially or completely reversible if the ischemic period was 5 minutes, whereas delayed degeneration occurred in the pyramidal cells of the medial CA1 region after reperfusion for 48 hours (delayed neuronal death). After 10 minutes' ischemia and subsequent reperfusion, delayed neuronal death extended from the medial to the lateral CA1 region; the ischemic and postischemic lesions in the cerebral cortex, thalamus, and caudoputamen also expanded during reperfusion. Our investigation demonstrates that selective vulnerability existed in global cerebral ischemia as in incomplete or regional ischemia and suggests that neurons in many areas of the brain possessed the potential for recovery, progressive deterioration, and even delayed neuronal death depending on the severity and duration of cerebral ischemia.

Immunohistochemical investigation of regional cerebral ischemia in the gerbil: occlusion of the posterior communicating artery.

Evolution, progression and recovery of neural damage during and following cerebral ischemia were investigated in the gerbil after occlusion of a posterior communicating artery and by using the immunohistochemical reaction for tubulin and creatine kinase BB-isoenzyme which are enriched in the neuronal structure and the reaction for astroprotein which is specific for astrocytes. The transcardiac perfusion study with India ink revealed marked hypoperfusion diffusely in the hippocampus and moderately in the thalamus on the occluded side. The earliest immunohistochemical lesion, manifested as loss of the reaction for tubulin and creatine kinase BB-isoenzyme in dendrites and nerve cell bodies, was found in the CA1 and CA2 region of the hippocampus after ischemia for 4 min, while it took 10 min before the earliest lesion became visible in the ventral nucleus of the thalamus and it took over 1 h before scattered lesions evolved in granular cells of the dentate gyrus. The staining with hematoxylin-eosin was much less sensitive in detection of early ischemic lesions. After re-establishment of blood flow to the posterior communicating artery, the ischemic lesions which were visualized with the reaction for tubulin or creatine kinase BB-isoenzyme disappeared or reduced the size, if the ischemic period was brief. Beyond a certain ischemic period, the lesion expanded further during the early postischemic period. The reaction for astroprotein visualized reactive astrocytes even in the area without any abnormalities with other reactions, an evidence of subtle ischemic insults.

Immunohistochemical investigation of cerebral ischemia during recirculation.

Immunohistochemical methods for the determination of tubulin, creatine kinase BB-isoenzyme, and astroprotein-glial fibrillary acidic protein were used to investigate recovery of the ischemic lesion after temporary occlusion of a common carotid artery in the gerbil and the evolution of the postischemic lesion following reperfusion. One group of gerbils was followed from 15 minutes to one month after an ischemic period of 30 minutes, and another group was examined after 7 days following an ischemic period of 5 to 30 minutes. It was found that the postischemic lesion, visualized as loss of the immunohistochemical reaction for tubulin and creatine kinase BB-isoenzyme, evolved within 60 minutes after reperfusion in the hippocampus and cerebral cortex and within 3 hours in the caudoputamen and thalamus. Resolution of the preexisting ischemic lesion was possible only after an ischemic period of less than 10 minutes in the cerebral cortex and caudoputamen and less than 15 minutes in the thalamus. In the CA1-CA2 region of the hippocampus, the ischemic lesion already existed after an ischemic period of 5 minutes and was mostly irreversible. The immunohistochemical method of testing for different cellular and subcellular components was very useful for investigation of cerebral ischemia and may also be advantageous for investigation of other pathophysiological conditions of the nervous system.

Immunohistochemical investigation of cerebral ischemia in gerbils.

Experimental cerebral ischemia was produced in gerbils by occlusion of the right common carotid artery in the neck. The evolution of the ischemic lesions was followed from five minutes to six hours by using the immunohistochemical techniques for tubulin and creatine kinase BB-isoenzyme. The earliest lesion was found in the subiculum-CA1 and CA2 regions of the hippocampus in five minutes. There was loss of staining in the apical dendrites and perikarya of the pyramidal cells. The earliest lesion in the cerebral cortex, visible in ten minutes, was a laminar loss of staining for tubulin. Evolution of the ischemic lesions in the thalamus and caudoputamen was delayed. However, in two hours widespread ischemic lesions were seen there. Evolution of the ischemic lesions was slightly slower with the reaction for creatine kinase BB-isoenzyme as compared to the reaction for tubulin, but was far more sensitive than hematoxylin-eosin staining. The distribution of ischemic lesions detected by the immunohistochemical method compared to ischemic areas detected by an India ink perfusion study suggested that both the extent of regional ischemia and regional difference in tissue vulnerability were contributing factors for the emergence of early ischemic lesions. The mechanism for prompt disappearance of the immunohistochemical reaction for tubulin is not clear, but the present investigation demonstrates the usefulness of the immunohistochemical technique for detecting early ischemic lesions and provides a possible biochemical mechanism for cellular damage after ischemic insults.

Immunohistochemical localization of creatine kinase BB-isoenzyme and tubulin in gerbil brain.

Cellular distribution of creatine kinase BB-isoenzyme and tubulin was investigated immunohistochemically with tissue sections from various areas of the gerbil brain. Both the reaction for creatine kinase BB-isoenzyme and tubulin visualized nerve cell bodies, dendrites and axons. While the reaction for tubulin was similar between nerve cell bodies and their dendrites, the reaction for creatine kinase BB-isoenzyme tended to be more intense in dendrites. While the reaction for tubulin could be seen in nerve cell bodies of different sizes, the reaction for creatine kinase BB-isoenzyme was more intense in larger neurons. Both the reaction for creatine kinase BB-isoenzyme and tubulin visualized astrocytic cytoplasm but only in the corpus callosum and in the white matter of limited areas. Visualization of dendrites to their peripheries with the reaction for creatine kinase BB-isoenzyme may indicate participation of this isoenzyme in maintainance of high energy-phosphates locally within dendrites. A combination of the immunohistochemical procedure for creatine kinase BB-isoenzyme and tubulin may be very useful for demonstration of various pathophysiological states of the central nervous system which affect dendrites.