Rituximab treatment did not aggravate ongoing progressive multifocal leukoencephalopathy in a patient with multiple sclerosis.

A multiple sclerosis (MS) patient developed progressive multifocal leukoencephalopathy (PML) after 43 months of natalizumab treatment. New clinical and magnetic resonance imaging (MRI) findings were initially misinterpreted as breakthrough MS disease activity and natalizumab treatment was replaced by rituximab treatment. The patient had a single infusion of rituximab 1000 mg before a definite PML diagnosis was confirmed. Despite undetectable levels of B-cells, JC virus DNA became undetectable in the cerebrospinal fluid by quantitative polymerase chain reaction. The patient partially recovered without any clinical or MRI signs of new MS activity. These findings suggest that B-cell depletion in a non-immune compromised individual did not prevent the patient from clearing the JC virus infection.

NMDA Receptor-dependent increase of cerebral glucose utilization after hypoxia-ischemia in the immature rat.

Post-treatment with the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 reduces hypoxic-ischemic brain injury in immature animals. To elucidate possible mechanisms, cerebral glucose utilization (CMRglc) and cerebral blood flow (CBF) were measured 1-5 h after hypoxia-ischemia and administration of MK-801 in 7-day-old rats. After 100 min of unilateral hypoxia-ischemia, half of the pups were injected with MK-801. CMRglc was assessed by the [14C]deoxyglucose (2-DG) method. The brains were analyzed either by autoradiography or for energy metabolites and chromatographic separation of 2-DG-6-phosphate and 2-DG. CBF was measured by the autoradiographic [14C]iodoantipyrine method. Mean CMRglc in the cerebral cortex was increased ipsilaterally after hypoxia-ischemia to 15 +/- 3.3 mumol 100 g-1 min-1 (p < 0.01) and areas with CMRglc > 20 mumol 100 g-1 min-1 amounted to 8.0 +/- 7.7 mm2 in the ipsilateral hemisphere compared with 1.2 +/- 1.6 mm2 contralaterally (p < 0.001). Treatment with MK-801 decreased CMRglc bilaterally (p < 0.05) and reduced ipsilateral areas with increased CMRglc by 64% (p < 0.01). CBF was unaltered after hypoxia-ischemia and by MK-801 treatment. In conclusion, regional glucose hyperutilization in the parietal cortex after hypoxia-ischemia was attenuated by MK-801; this may have relevance to the neuroprotective effect of NMDA-receptor antagonists in this model.

Temporal changes of regional glucose use, blood flow, and microtubule-associated protein 2 immunostaining after hypoxia-ischemia in the immature rat brain.

In a situation with normal CBF and without increased energy utilization, increased glucose utilization (CMRglc) can be a sign of impaired mitochondrial metabolism, which may be an early step in the injury cascade during reperfusion after hypoxia-ischemia (HI). Seven-day-old rats underwent unilateral carotid artery ligation and 70 minutes of HI. At 3, 6, 12, 24, and 48 or 72 hours after the insult, the CMRglc was measured by the 2-deoxyglucose method, and CBF by the iodoantipyrine method. These were compared with hematoxylin-eosin staining and microtubule-associated protein 2 (MAP 2) immunostaining in adjacent sections. In the ipsilateral hemisphere, there appeared regions with increased CMRglc compared with the contralateral hemisphere 3 to 12 hours after HI that also showed partial loss of MAP 2 immunostaining and early ischemic changes. These areas receded, leaving central glucose hypoutilizing areas with complete loss of MAP 2 immunostaining and histologic infarction, surrounded by only a rim of tissue with increased CMRglc. At 24 and 72 hours after the insult, no regions with increased CMRglc remained. Despite loss of MAP 2 immunostaining and histologic signs of infarction at 24 hours, cortical CBF was not reduced until 48 hours after HI, whereas the CBF in the caudate-putamen already was decreased compared with the contralateral side at 3 hours after HI. In conclusion, early reperfusion is characterized by glucose hyperutilizing areas in the cerebral cortex, followed by a secondary phase with low CMRglc and infarction.

Mitochondrial function and energy metabolism after hypoxia-ischemia in the immature rat brain: involvement of NMDA-receptors.

Treatment after hypoxia-ischemia (HI) in immature rats with the N-methyl-D-aspartate receptor (NMDAR) antagonist dizocilpine maleate (MK-801) reduces areas with high glucose utilization and reduces brain damage. The object was to study the metabolic effects of MK-801 treatment after HI. Seven-day-old rats were randomized to the following groups: non-HI, HI, or HI plus MK-801 (0.5 mg/kg immediately after HI). In the parietal cortex, the mitochondrial respiration was measured in homogenates 1 to 4 hours, and the energy metabolites at 3 and 8 hours after HI. The energy use was calculated from changes in energy metabolites after decapitation at 3 hours after HI. State 3 respiration was reduced by 46%, 32%, and 25% after HI compared with non-HI with pyruvate plus malate, glutamate plus malate, or glutamate plus succinate as substrates, respectively. Uncoupler-stimulated but not state 4 respiration was similarly reduced. The MK-801 augmented pyruvate plus malate-supported state 3 respiration after HI by 42%. The energy utilization was not affected by HI but was reduced by MK-801 treatment in the ipsilateral cortex from 4.6 +/- 2.3 to 2.6 +/- 1.8 micromol high-energy phosphate bond/min/g. The levels of ATP and phosphocreatine did not differ between the HI and HI plus MK-801 groups at 3 hours, but were lower in the HI than in the HI plus MK-801 group at 8 hours after HI. In conclusion, treatment with MK-801 reduced energy utilization and improved mitochondrial function and energy status after HI, suggesting a linkage between NMDAR activation and impaired energy metabolism during reperfusion.

Rhombomeric organization of vestibular pathways in larval frogs.

Rhombencephalic subnuclei and projection pathways related to vestibular function were mapped in larval ranid frogs. The retention of overt postembryonic rhombomeres (r) allowed direct visualization of the locations of neurons retrogradely labeled with fluorescent dextran amines from the midbrain oculomotor complex, cerebellum, vestibular nuclei, and spinal cord. Oculomotor projecting vestibular neurons were mainly located in bilateral r1/2, ipsilateral r3, and contralateral r5-8, and spinal projecting vestibular neurons mainly in ipsilateral r4 and contralateral r5. Vestibular commissural neurons were located in r1-3 and r5-7 and were largely excluded from r4. Cerebellar projecting neurons included contralateral inferior olivary neurons in r8 and vestibular neurons in bilateral r6/7 and contralateral r1/2. Mapping these results onto adult anuran vestibular organization indicates that the superior vestibular nucleus derives from larval r1/2, the lateral vestibular nucleus from r3/4, and the major portions of the medial and descending vestibular nuclei from r5-8. The lateral vestibulospinal tract projects from an origin in r4, whereas a possible ascending tract of Deiters arises in r3. Rhombomere 5 contains a nuclear group that appears homologous to the tangential nucleus of fish, reptiles, and birds and thus likely serves gravistatic and linear vestibulomotor reflexes. Comparisons between frogs and other vertebrates suggest that vestibular neurons performing similar computational roles during head movements originate from the same segmental locations in different species.

Neonatal cerebral hypoxia-ischemia: the effect of adenosine receptor antagonists.

The effects of nonselective (theophylline), A1-(DPCPX) or A2A-selective (SCH 58261) adenosine receptor antagonists administered before or after neonatal hypoxia-ischemia (HI) were studied on the extent of brain injury in 7-day-old rats evaluated after 14 days. A possible effect of theophylline (20 mg/kg) on expression of immediate early genes was studied with in situ hybridization. Theophylline (20, 30 or 60 mg/kg) given prior to HI reduced brain damage by 48% (P < 0.001), 36% (P < 0.01) and 34% (P < 0.05), respectively, compared to control rats. This effect was not explained by changes in temperature, cerebral blood flow, blood gas/acid base status or blood glucose during the insult. Theophylline enhanced the upregulation of c-fos and NFGI-A during reperfusion but did not prevent the decrease in adenosine A1 receptor mRNA. Posttreatment with SCH 58261 (0.2 or 2 mg/kg) reduced brain damage by 19% (P < 0.05) and 14% (NS), respectively, compared to control rats which was unrelated to the core temperature. DPCPX (2 or 10 mg/kg) had no effect on the development of brain injury. In conclusion, nonselective and A2A adenosine receptor antagonists reduced brain injury in a model of HI in immature animals.

Otolith ocular reflex function of the tangential nucleus in teleost fish.

In teleost fish, the tangential nucleus can be identified as a compact, separate cell group lying ventral to the VIIIth nerve near the middle of the vestibular complex. Morphological analysis of larval and adult hindbrains utilizing biocytin and fluorescent tracers showed the tangential nucleus to be located entirely within rhombomeric segment 5 with all axons projecting into the contralateral MLF. Combined single-cell electrophysiology and morphology in alert goldfish found three classes of neurons whose physiological sensitivity could be readily correlated with rotational axes about either the anterior (45 degrees), posterior (135 degrees), or horizontal (vertical axis) semicircular canals. Tangential neurons could be distinguised from those in semicircular-canal specific subnuclei by an irregular, spontaneous background of 10-15 sp/s and sustained static sensitivity after +/- 4 degrees head displacements. Each axis-specific tangential subtype terminated appropriately onto oculomotor subnuclei responsible for either vertical, torsional, or horizontal eye movements and, in a few cases, axon collaterals descended in the MLF toward the spinal cord. We hypothesize, therefore, that the tangential nucleus consists of 3 axis-specific phenotypes that process gravitoinertial signals largely responsible for controlling oculomotor function, but that also in part, maintain body posture.

Is MK-801 neuroprotection mediated by systemic hypothermia in the immature rat?

Hypothermia after hypoxia-ischaemia (HI) confounds the interpretation of the effects of neuroprotective drug intervention. The effect of 0.5 mg/kg of dizocilpine (MK-801) administered after HI on rectal temperature at 2-36 h and on brain damage 2 weeks after the insult was evaluated in the immature rat. In pups kept at an ambient temperature of 21 degrees C, MK-801 lowered the temperature by 1.1 degrees C and reduced the brain damage by 45%. In pups held at an ambient temperature of 33 degrees C, MK-801 treatment afforded a 34% reduction of brain damage without lowering the rectal temperature. In conclusion, the neuroprotection offered by MK-801 does not depend on systemic hypothermia in this model.

Microglia activation after neonatal hypoxic-ischemia.

The inflammatory response following hypoxic-ischemia (HI) in the neonate is largely unknown. Presently, the expression of microglial antigens and the beta-amyloid precursor protein (APP) were studied in relation to a dendrosomatic marker of neuronal injury (microtubule associated protein II; MAP II). HI was induced in 7-day-old rats by the combined unilateral carotid ligation and hypoxia. The pups (n = 23) were perfusion fixed 2-3 h, 24 h, 2-4 days and 14 days after HI and compared to sham-operated controls (n = 6). Antibodies were used for detection of the major histocompatibility complex II (OX-6), major histocompatibility complex I (OX-18) and complement receptor type 3 (OX-42), APP (APP 676-695) and MAP II (monoclonal MAP II) antigens. There was a transient APP expression 2-3 h after HI. A slight increase of microglial antigens (OX-18) was seen in the white matter 2 h after HI followed by a marked increase of OX-18, OX-6, OX-42 antigens 24 h-3-4 days in most injured regions with exception of the thalamus where a delayed (14 days) microglial response was seen. The latter event was parallelled by a delayed loss of MAP II. In conclusion, intense microglial expression occurs after neonatal HI either with an acute or delayed time-course depending on brain region.

Hypoxic-ischemic injury in the neonatal rat brain: effects of pre- and post-treatment with the glutamate release inhibitor BW1003C87.

In a model of perinatal hypoxia-ischemia (HI) we examined the neuroprotective efficacy of pre- and post-treatment with the glutamate release inhibitor BW1003C87 [5-(2,3,5-trichlorophenyl)-2,4-diamino-pyrimidine). Ipsilateral brain damage developed in 99% of rat pups subjected to HI (unilateral common carotid artery ligation and 100 min of 7.7% oxygen exposure) with a 26 +/- 16% (mean +/- S.D.) weight deficit of the damaged hemisphere 2 weeks after the insult. Pre-treatment with BW1003C87 (10 mg/kg intraperitoneally) reduced the brain damage by 46% (P < 0.05). A higher dose (20 mg/kg) of pre-treatment was not tolerated. Administration of BW1003C87 did not affect the rectal temperature of the rats. Post-treatment with BW1003C87 (10-30 mg/kg) offered no neuroprotection in this model. In conclusion, there was a neuroprotective effect from pre- but not post-treatment with BW1003C87 in this model, supporting the concept that intra-ischemic excitatory amino acid release is important for development of brain damage. The lack of post-treatment effect indicates that BW1003C87 did not attenuate deleterious EAA cycling during reflow in the neonatal brain.

Impairment of mitochondrial respiration after cerebral hypoxia-ischemia in immature rats: relationship to activation of caspase-3 and neuronal injury.

Mitochondrial damage may play a key role in the development of necrotic and apoptotic hypoxic-ischemic (HI) brain damage. It has previously been shown that mitochondrial respiration is depressed in the cerebral cortex after HI in neonatal animals. The aim of the present study was to further characterize the time course of the mitochondrial impairment during reperfusion and the correlation between the respiratory control ratio and brain injury and activation of caspase-3. Rat pups were subjected to unilateral carotid artery ligation and exposed to hypoxia (7.7% oxygen). Mitochondrial respiration was measured 0-72 h after HI in a mitochondrial fraction isolated from cerebral cortex. Microtubule associated protein-2 (MAP2) and caspase-3 were analyzed with immunoblotting in cerebral cortex homogenates. In addition, the time course of caspase-3 activation was measured as DEVD cleavage. The mitochondrial respiratory control ratio in cerebral cortex decreased immediately after HI followed by a partial recovery at 3-8 h. Thereafter, a secondary drop occurred with a minimum reached at 24 h of reperfusion. The secondary loss of respiratory function was accompanied by depletion of MAP2, cleavage of caspase-3 and an increased caspase-3 -like activity at 3-24 h after the insult. In conclusion, the primary phase of mitochondrial dysfunction was paralleled by a moderate decrease of MAP2 and a limited activation of caspase-3. The secondary mitochondrial impairment was associated with neuronal injury and pronounced activation of caspase-3.

Hypoxia-ischaemia model in the 7-day-old rat: possibilities and shortcomings.

The Levene model in 7-day-old rats is the most often used model of hypoxia-ischaemia (HI) in immature animals. The rat central nervous system is immature at birth and corresponds neurodevelopmentally to the term human infant during the second postnatal week. The Levene model of HI differs from clinical asphyxia with respect to the unilateral distribution of brain injury and lack of multi-organ dysfunction. Furthermore, it does not allow cardiovascular monitoring or repeated blood sampling. On the other hand, the progressive nature of HI bears many similarities to birth asphyxia with regard to blood flow changes and cellular metabolic derangements. The model is well characterized, easy to carry out and the low cost allows inclusion of a sufficient number of animals for dose-response evaluation of neuroprotective agents. In addition, it provides the unique opportunity of long-term evaluation of neuropathological and functional outcome.

A hindbrain segmental scaffold specifying neuronal location in the adult goldfish, Carassius auratus.

The vertebrate hindbrain develops as a series of well-defined neuroepithelial segments or rhombomeres. While rhombomeres are visible in all vertebrate embryos, generally there is not any visible segmental anatomy in the brains of adults. Teleost fish are exceptional in retaining a rhombomeric pattern of reticulospinal neurons through embryonic, larval, and adult periods. We use this feature to map more precisely the segmental imprint in the reticular and motor basal hindbrain of adult goldfish. Analysis of serial sections cut in three planes and computer reconstructions of retrogradely labeled reticulospinal neurons yielded a segmental framework compatible with previous reports and more amenable to correlation with surrounding neuronal features. Cranial nerve motoneurons and octavolateral efferent neurons were aligned to the reticulospinal scaffold by mapping neurons immunopositive for choline acetyltransferase or retrogradely labeled from cranial nerve roots. The mapping corresponded well with the known ontogeny of these neurons and helps confirm the segmental territories defined by reticulospinal anatomy. Because both the reticulospinal and the motoneuronal segmental patterns persist in the hindbrain of adult goldfish, we hypothesize that a permanent "hindbrain framework" may be a general property that is retained in adult vertebrates. The establishment of a relationship between individual segments and neuronal phenotypes provides a convenient method for future studies that combine form, physiology, and function in adult vertebrates.

Conservation of neuroepithelial and mesodermal segments in the embryonic vertebrate head.

The organization of embryonic efferent cranial nerves is addressed here by interspecies comparison of segmentally patterned neuromeres, efferent neuronal populations and early mesodermal sources of target muscles. The segmental constancy of these three structural patterns is evaluated for elasmobranch, teleost, reptile, bird and mammal embryos and compared with the segmentally restricted expression patterns of Hox genes. A conserved series of hindbrain neuroepithelial segments (rhombomeres) is present in all of these taxa. Dye-labeling experiments demonstrate that the segmental locations of efferent neurons projecting through individual cranial nerves are likewise highly conserved. Notable segmental variation is however shown in the location of the VI and IX-XII motoneurons, suggesting the likelihood of homeotic-like changes in relations between rhombomere and neuronal 'identity' during vertebrate evolution. Since experimentally induced shifts in expression borders of Hox genes appear to be correlated with alterations in segment identity and/or neuronal phenotype, the need for further examination of segmental locations of specific neuronal groups and the segmental expression patterns of Hox genes between species is emphasized. Comparison of early cranial mesodermal subdivisions in elasmobranchs with descriptions of somitomeres in amniotes suggests that a series of axially unique mesodermal populations may also be conserved throughout vertebrates. The possibility is raised that common mechanisms of axial specification may underlie the initial appearance of segmental patterning in both neural and mesodermal layers during gastrulation. Implications of these conserved patterns for understanding the phylogenetic origin of the vertebrate head are briefly discussed.

Cerebral hypoxia-ischemia in immature rats: involvement of mitochondrial permeability transition?

The aim of this study was to evaluate the involvement of mitochondrial membrane permeability transition (MPT) after hypoxia-ischemia (HI) in 7-day-old rats. [14C]2-deoxyglucose (DOG) was administered to controls, and at various time points after HI. MPT in the cerebral cortex was measured as entrapment of DOG-6-P in mitochondria. Another group of rats was treated with the MPT inhibitor cyclosporin A (CsA; 10-50 mg/kg i.p.) or vehicle before and after HI, and the effect on brain injury and mitochondrial respiration was evaluated. A significant increase in DOG-6-P entrapment in mitochondria indicated that MPT occurred in two phases: a primary MPT after 0-1.5 h and a secondary MPT after 6.5-8 h of reperfusion. However, CsA did not affect brain injury or mitochondrial respiration. The data suggest that MPT occurred after HI but does not provide evidence for its involvement in the development of injury.

Hypoxia-ischemia in the neonatal rat brain: histopathology after post-treatment with NMDA and non-NMDA receptor antagonists.

In a model of perinatal hypoxic-ischemic brain damage, we examined the neuroprotective efficacy of posttreatment with the NMDA receptor antagonist MK-801 and the AMPA receptor antagonist NBQX. Unilateral brain damage developed in 95% of rat pups subjected to hypoxia-ischemia with a 27.8 +/- 1.2% weight deficit of the damaged hemisphere. MK-801 in doses of 0.3 and 0.5 mg/kg i.p. reduced the brain damage by 61% (p < 0.001) and 43% (p < 0.001), respectively. A higher dose of MK-801 (0.75 mg/kg) did not offer neuroprotection. Treatment with NBQX (40 mg/kg) reduced the hemispheric lesion by 28% (p < 0.05). In conclusion, posttreatment with both NBQX and low doses of MK-801 reduced perinatal brain damage. The NMDA receptor antagonist offered stronger neuroprotection which is in agreement with a proposed NMDA receptor hyperactivity around postnatal day 7 in rats.

Magnesium and the N-methyl-D-aspartate receptor antagonist dizocilpine maleate neither increase glucose use nor induce a 72-kilodalton heat shock protein expression in the immature rat brain.

In adult rats N-methyl-D-aspartate receptor (NMDAR) antagonists increase glucose use and induce a 72-kD heat shock protein (HSP72) expression in limbic system areas that later undergo neuronal necrosis, which have limited the clinical development of these drugs. Dizocilpine maleate (MK-801) and magnesium sulfate (MgSO4) reduce hypoxic-ischemic brain injury in immature animals, but the effects on HSP72 expression and glucose use are unknown. Seven-day-old rats received injections of either vehicle (control), 0.5 or 1.0 mg/kg MK-801, or 2 or 4 mmol/kg MgSO4. Glucose utilization was measured with the deoxyglucose method, 30 min, 48 h, and 4 d after injection. HSP72 immunostaining was evaluated 4 or 24 h after injection. Both doses of MK-801 and 4 mmol/kg MgSO4 induced a temporary decrease in glucose use in the posterior cingulate and retrosplenial cortex, the CA1 and CA3 subfields of the hippocampus, the caudoputamen, and the parietal cortex. Doses of 2 mmol/kg MgSO4 did not affect glucose use in any structure. Neuronal HSP72 expression was not found in any drug-treated rats. In conclusion, neither MK-801 nor MgSO4 increased glucose use in the limbic system and did not induce HSP72 expression, suggesting that NMDAR antagonists lack direct neurotoxicity in the immature brain.

Imaging of multicellular large-scale rhythmic calcium waves during zebrafish gastrulation.

Oscillations of cytosolic free calcium levels have been shown to influence gene regulation and cell differentiation in a variety of model systems. Intercellular calcium waves thus present a plausible mechanism for coordinating cellular processes during embryogenesis. Herein we report use of aequorin and a photon imaging microscope to directly observe a rhythmic series of intercellular calcium waves that circumnavigate zebrafish embryos over a 10-h period during gastrulation and axial segmentation. These waves first appeared at about 65% epiboly and continued to arise every 5-10 min up to at least the 16-somite stage. The waves originated from loci of high calcium activity bordering the blastoderm margin. Several initiating loci were active early in the wave series, whereas later a dorsal marginal midline locus predominated. On completion of epiboly, the dorsal locus was incorporated into the developing tail bud and continued to generate calcium waves. The locations and timing at which calcium dynamics are most active appear to correspond closely to embryonic cellular and syncytial sites of known morphogenetic importance. The observations suggest that a panembryonic calcium signaling system operating in a clock-like fashion might play a role during vertebrate axial patterning.