Fabián Bernal

Immunohistochemical analysis of anti-Hu-associated paraneoplastic encephalomyelitis

Abstract. The precise immune mechanisms of neuronal death in anti-Hu-associated paraneoplastic encephalomyelitis (PEM) are unclear. We performed an immunohistochemical study on postmortem brain tissue from 11 patients with anti-Hu-associated PEM to further characterize the immune reaction and to ascertain possible mechanisms of neuronal death. To analyze inflammatory infiltrates, antibodies against lymphocyte subpopulations (CD3, CD20, CD4, CD8), macrophage and activated microglia (CD68), major histocompatibility complex (MHC) classes I and II (HLA-ABC and HLA-DR), and the intercellular adhesion molecules (ICAM) -1 and -3 were used. Cell death mechanisms were defined using antibodies against the cytotoxic protein TIA-1, the C9neo component of complement, the Fas receptor (CD95) and its ligand, the apoptosis effector activated caspase-3, and the apoptosis inhibitor Bcl-2. A great number of T cells expressing the cytotoxic protein TIA-1 was observed, mainly in clusters around neurons. ICAM-1 immunoreactivity was increased in the neuropil and reactive astrocytes in areas of inflammation within the central nervous system and in satellite cells of pathological dorsal root ganglia surrounding apparently normal sensory neurons. By contrast, Fas, FasL, C9neo, and activated caspase-3 immunoreactivities were negative in pathological areas. Bcl-2 immunoreactivity was found in satellite cells, but not in sensory neurons of normal and pathological dorsal root ganglia. Our data point out to an induction of a cytotoxic, non-apoptotic, neuronal death in anti-Hu-associated PEM. The increased ICAM-1 immunoreactivity may favor the infiltration of lymphocytes in the pathological areas.

Differential vulnerability of hippocampus, basal ganglia, and prefrontal cortex to long-term NMDA excitotoxicity.

In human brain, nonartherosclerotic calcification is associated with normal aging and several pathological conditions without any clear significance. In all situations, calcification appears predominantly in the basal ganglia, but is also frequent in the hippocampus and cerebral cortex. alpha-Amino-(3-hydroxi-5-methyl-4-isoxazol-4-il)-propionic acid-induced lesion of the globus pallidus is associated in rats with the formation of calcium deposits similar to those observed in the human brain. To determine whether direct neuronal activation may induce calcification, N-methyl-d-aspartate (NMDA) was microinjected in rat hippocampus, globus pallidus, and lateral prefrontal cortex. Two months later, neuronal death was associated with calcium deposits that were characterized in terms of distribution and size. A unique population of deposits was present in the hippocampus and prefrontal cortex, whereas in the globus pallidus two main groups could be differentiated. Calcification was always associated with a significant microglial reaction as shown by the peripheral benzodiazepine receptor autoradiography. Monoamine oxidase B autoradiography, reflecting the astroglial reaction, was also significantly increased. Our results provide evidence that acute NMDA neuronal activation leads with time to calcification associated with a glial reaction and indicate that nonartherosclerotic calcification in the human brain may develop from an acute NMDA receptor activation. A key role of the metabotropic mGluR1 receptor is also suggested.

Basal ganglia calcification induced by excitotoxicity: an experimental model characterised by electron microscopy and X-ray microanalysis

Abstract Activation of glutamate receptors induces an excitotoxic neurodegenerative process characterised in some brain areas by the formation of calcium precipitates. To examine the pathogenesis of basal ganglia calcification (BGC), an improved procedure of X-ray microanalysis was used to study experimental excitotoxic calcification in the rat. Three weeks after injection of ibotenic acid (IBO) in the rat basal forebrain, calcified inclusions within hypertrophied astrocytes were characterised. They appeared to form part of a filamentous structure localised in the cytoplasm in association with normal mitochondria and other organelles. Larger inclusions were surrounded by reactive microglia. The main inorganic components in these deposits were Ca and P, frequently accompanied by S, Al, Si and K. The shape and Ca/P molar ratio of the large deposits (> 10 μm) indicate that they may be biological apatites. Aluminosilicates were detected as small deposits (< 4 μm) free of other mineral constituents. To our knowledge this is the first report showing that IBO lesion induces brain accumulation of aluminosilicates similar to that described in Alzheimer’s or Fahr’s patients. Our data indicate that precipitation of Ca and Al may reduce their IBO-induced increased concentration. In conclusion, the experimental model and the improved efficiency of X-ray analysis described may help us to understand the pathogenesis of BGC.

Excitatory amino acids and neurodegeneration: a hypothetical role of calcium precipitation.

Activation of excitatory amino acid (EAA) receptors can induce neurodegeneration by two major mechanisms of excitotoxicity, one related to the influx of Na+, Cl− and water, and the other to the increase in intracellular calcium concentration ([Ca2+]i). Thus, acute microinjection of EAAs in several areas of the central nervous system (CNS) has been used to produce neurodegenerative models. We studied the excitotoxic pattern associated with acute microinjection of AMPA in rat hippocampus, medial septum‐diagonal band of Broca (MS‐DBB), prefrontal cortex and retina. In all cases progressive neuronal loss, glial reaction and development of intra‐ and extracellular calcium concretions were observed. However, a CNS‐area differential vulnerability was revealed, as shown by the specific atrophy of MS‐DBB and its limited calcification. Whether calcium deposits are a defensive mechanism against the massive increment of free cytoplasmatic calcium is discussed on the basis of ultrastructural data and previous results.

Perinatal human hypoxia-ischemia vulnerability correlates with brain calcification.

Deregulation of intracellular calcium homeostasis is widely considered as one of the underlying pathophysiological mechanisms of hypoxic-ischemic brain injury. Whether this alteration can result in cerebral calcification was investigated in basal ganglia, cerebral cortex, and hippocampus of human premature and term neonates together with glial reaction. In all samples nonarteriosclerotic calcifications were observed, their number and size were area-specific and increased in term neonates. Basal ganglia always presented the highest degree of calcification and hippocampus the lowest, located mainly in the CA1 subfield. In all cases, neuronal damage was associated with astroglial reaction and calcium precipitates, with microglial reaction only in basal ganglia and cerebral cortex, and argues for the participation of excitatory amino acid receptors in hypoxia-ischemia damage. These data correlate with hypoxia-ischemia vulnerability in the perinatal period. The clinical relevance of these precipitates and the neuroprotective interest of non-NMDA receptor manipulation are discussed in the light of our results.

Heterogeneity between hippocampal and septal astroglia as a contributing factor to differential in vivo AMPA excitotoxicity

Astroglial participation in the regional differences of vulnerability to α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA)‐induced neurodegeneration was investigated in the rat hippocampus and medial septum using L‐α‐aminoadipate (α‐AA) as a specific astroglial toxin. α‐AA was microinjected in the hippocampus and the medial septum and a time‐course study was carried out between 2 hr and 3 days. When compared to controls, microinjection of α‐AA in the hippocampus induced within 3 days a reversible loss of glial fibrillary acidic protein (GFAP) immunostaining and a microglial reaction without any neuronal loss, whereas in the medial septum it caused no effects on astroglial, microglial, or neuronal populations. Differences in hippocampus and medial septum vulnerability were also evidenced when α‐AA was co‐injected with AMPA and neurodegeneration was assessed in terms of neuronal loss, glial reactions, calcification, and atrophy of the area. In the hippocampus, α‐AA increased AMPA excitotoxicity with marked disorganization of all hippocampal subfields, increased neuronal loss, a more important astroglial reaction, a larger area of microgliosis, and a greater abundance of calcium deposits. By contrast, in the medial septum α‐AA did not modify any parameter of the AMPA‐induced lesion. In conclusion, the presence of different astroglial populations in hippocampus and medial septum results in a different participation to AMPA excitotoxicity that may determine, at least in part, the specific regional vulnerability to neurodegeneration.

Similar calcification process in acute and chronic human brain pathologies

Cellular microcalcification observed in a diversity of human pathologies, such as vascular dementia, Alzheimers disease, Parkinsons disease, astrogliomas, and posttraumatic epilepsy, also develops in rodent experimental models of central nervous system (CNS) neurodegeneration. Central to the neurodegenerative process is the inability of neurons to regulate intracellular calcium levels properly, and this is extensible to fine regulation of the CNS. This study provides evidence of a common pattern of brain calcification taking place in several human pathologies, and in the rat with glutamate‐derived CNS lesions, regarding the chemical composition, physical characteristics, and histological environment of the precipitates. Furthermore, a common physical mechanism of deposit formation through nucleation, lineal growth, and aggregation is presented, under the modulation of protein deposition and elemental composition factors. Insofar as calcium precipitation reduces activity signals at no energy expense, the presence in human and rodent cerebral brain lesions of a common pattern of calcification may reflect an imbalance between cellular signals of activity and energy availability for its execution. If this is true, this new step of calcium homeostasis can be viewed as a general cellular adaptative mechanism to reduce further brain damage.

Age-related resistance to ?-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-induced hippocampal lesion

This study compares the effects of acute α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA) administration in the hippocampus in adult (3 months) and middle‐aged (15 months) rats at 15 days postinjection. Injection of 1 and 2.7 mM AMPA produced dose‐dependent neurodegeneration, assessed by Nissl staining; a glial reaction shown by glial fibrillary acidic protein immunocytochemistry; and calcification, revealed by alizarin red staining. Furthermore, at both doses, these alterations were significantly greater in 3‐month‐old rats. Finally, at AMPA 2.7 mM, no significant changes in the density of hippocampal parvalbumin‐ or calbindin‐immunoreactive neurons or in choline acetyltransferase, glutamate uptake, or GABA uptake activities were found in 15‐month‐old animals, whereas significant reductions in parvalbumin (–76%) and calbindin (–32%) immunostaining and in GABA uptake (–27%) were observed in 3‐month‐old animals compared to the respective sham‐operated or control animals. In conclusion, this study clearly demonstrates that in rats the vulnerability of hippocampal neurons and the glial and calcification reactions to AMPA‐induced injury decreased with age between 3 and 15 months. Our results also indicate that hippocampal cholinergic, glutamatergic, and GABAergic systems show an adaptive response to excitotoxic damage in both adult and middle‐aged animals. Hippocampus 10:296–304, 2000

Nimodipine inhibits TMB-8 potentiation of AMPA-induced hippocampal neurodegeneration.

Human cerebral calcification has been related to deregulation of intracellular calcium homeostasis. In rat basal ganglia, nimodipine and TMB‐8, two commonly used calcium antagonists, worsen the chronic AMPA‐induced lesion, whereas only nimodipine potentiates calcification. To investigate whether similar effects are present in the hippocampus, AMPA dose–response and calcium movement blockade were performed. A dose‐related increase of both hippocampal lesion and calcification was evident in a saturable mode, mostly different from the continuous globus pallidus response previously observed. The value of 2.7 nmol AMPA, selected as yielding 60% of maximum calcification, was coinjected with nimodipine or/and TMB‐8 to determine their influence on tissue damage. TMB‐8 increased the AMPA lesion in terms of calcified area, and nimodipine reversed this increase, with no effect alone. These results, divergent from those for the globus pallidus, reveal differences in extra‐ and intracellular calcium movement between the two neurodegenerative processes. Future work focused on other brain areas is required to understand how control of calcium stores may influence neurodegenerative disease evolution.

Nimodipine and TMB-8 potentiate the AMPA-induced lesion in the basal ganglia.

Acute injection of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) into the rat globus pallidus leads to calcium precipitation, neuronal death and gliosis. In order to determine whether L-type calcium channels and/or release of Ca(2+) from intracellular stores contribute to the effects of AMPA, nimodipine and 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) were administered in combination with AMPA. Nimodipine, but not TMB-8, tended to exacerbate the calcification process initiated by AMPA; the AMPA/nimodipine/TMB-8 combination produced much more calcium deposition than AMPA (+62%, P<0.05). AMPA alone induced a slight but not significant astroglial reaction. Nimodipine slightly enhanced the astroglial reaction triggered by AMPA, whereas TMB-8 doubled it (P<0.001 versus AMPA). These data suggest that blockade of L-type calcium channels by nimodipine enhances calcium imbalance triggered by AMPA, and the calcium release from the endoplasmic reticulum does not participate in the AMPA-induced calcification.