Pontosubicular Neuron Necrosis Research Articles

Fetal Central Nervous System Injury in Third Trimester Stillbirth: A Clinicopathologic Study of 63 Cases

We report the neuropathologic findings and clinicopathologic associations in 63 3rd trimester singleton stillborn fetuses. All were ≥ 28 weeks estimated gestational age (EGA) with complete autopsies, including placental examination. Fetuses with chromosomal abnormalities, major congenital anomalies, and intrapartum demise were excluded. The cases were divided into those with abruption (n = 12) and those with unexplained fetal demise (n = 51). The latter group was then subdivided by gestational age with 3 subgroups (preterm 28 to < 32 weeks EGA (n = 16), preterm 32 to <37 weeks EGA (n = 13), and term 37-41 weeks EGA (n = 22). Each group was further subdivided as appropriate-for-gestational age/large-for-gestational age (AGA/LGA) or small-for-gestational age (SGA). Placental lesions were also evaluated and correlated with brain lesions. Established or recent injury involving gray or white matter was seen in 88% of the fetuses with unexplained demise versus 42% with abruption (P = 0.001). The most common form of brain injury was established gray matter damage, seen in 65% of the fetuses with unexplained demise versus 25% with abruption (P = 0.021), the most common pattern being established pontosubicular neuronal necrosis plus established neuronal necrosis in other sites. There was no significant difference in the frequency of brain injury between the SGA fetuses and AGA/LGA fetuses or between the unexplained stillbirth preterm and term subgroups, and there was no unequivocal correlation between placental lesions and brain lesions. Brain injury, most frequently established gray matter damage, is seen in the majority of stillborn infants with unexplained demise, indicating that the brain injury predates the period immediately before death.

A histopathological study of premature and mature infants with pontosubicular neuron necrosis: Neuronal cell death in perinatal brain damage

Perinatal hypoxic–ischemic brain damage is a major cause of neuronal and behavior deficits, in which the onset of injury can be before, at or after birth, and the effects may be delayed. Pontosubicular neuron necrosis (PSN) is one of perinatal hypoxic–ischemic brain injury and its pathological peculiarity is neuronal apoptosis. In this study, we investigated whether apoptotic cascade of PSN used a caspase-pathway or not, and whether hypoglycemia activated apoptosis or not. Sections of the pons of PSN with and without hypoglycemia were stained using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) and immunohistochemistry for glial fibrillary acidic protein (GFAP), Bcl-2, Bcl-x and activated caspase 3. Additionally, we performed immunoblot analysis of Bcl-2, Bcl-x and activated caspase 3. TUNEL-positive cell was closely associated with the presence of karyorrhexis. Under combination of karyorrhectic and TUNEL-positive cells, number of apoptotic cells in premature brains was significantly more than in mature brains. Hypoxic–ischemic brain injury was considered to easily lead to apoptosis in premature infants. Moreover, as this pathophysiology, caspase-pathway activation contributed to neuronal death from caspase-immunoexpression analyses. PSN with hypoglycemia showed large number of apoptotic cells and higher expression of activated caspase 3. The result may be more severe with the background of hypoglycemia and prematurity complicated by hypoxia and/or ischemia.

259 RBM3 Expression in Neonates with Pontosubicular Neuron Necrosis

Objective: Pontosubicular neuron necrosis (PSN) represents an age-specific response to severe hypoxic-ischemic injury (HII) occurring in human neonates but not in older children or adults. Histologically, PSN is characterised by acute neuronal death in the pontine nuclei and the hippocampal subiculum bearing the hallmarks of apoptosis. The expression of Rbm3, a glycine-rich RNA-binding protein, is enhanced under hypoxic conditions and independent of HIF (hypoxia inducible factor). It is well known, that proteins which are dependant on HIF, e.g. Erythropoetin play an important role in HII. This study aims to determine whether the HIF-independent activation of Rbm3 is also a significant factor in the pathogenesis of PSN. Methods: We have investigated the expression of Rbm3 in human autopsy material consisting of 12 PSN cases and 10 age-matched controls without PSN. Immunohistochemistry and double labeling for Rbm3 and the astrocyte marker glial fibrillary acid protein (GFAP), the microglia/macrophage specific marker KiM1P and the neuronal marker NeuN was performed on formalin-fixed, paraffin-embedded brain specimens.Results: In PSN cases and controls, mainly neuronal cells expressed Rbm3. The number of immunopositive cells was significantly increased (p=0.001) in PSN cases. Predominantly degenerating cells with signs of later apoptotic stages showed Rbm3 expression. In earlier stages of apoptosis immunopositivity for Rbm3 was increased compared to contols, but less prominent.Conclusion: In addition to HIF-dependant proteins, the induction of the HIF-independent protein RMB3 is observed in response to human hypoxic-ischemic injury.

Fas (CD95/Apo-1)/Fas ligand expression in neonates with pontosubicular neuron necrosis.

Pontosubicular neuron necrosis (PSN) represents an age-specific response to severe hypoxic-ischemic injury occurring in human neonates but not in older children or adults. Histologically, PSN is characterized by acute neuronal death in the pontine nuclei and the hippocampal subiculum bearing the hallmarks of apoptosis. In animal models of hypoxic-ischemic injury, induction of neuronal apoptosis can be triggered by Fas (CD95/Apo-1), a cell surface receptor of the tumor necrosis factor-alpha superfamily, which transduces apoptotic death signals when cross-linked by its natural ligand. Here, we have investigated the expression of Fas/Fas ligand in human autopsy material consisting of 13 PSN cases and 10 age-matched cases without PSN. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, immunohistochemistry, and double labeling for Fas/Fas ligand and the astrocyte marker glial fibrillary acid protein, the microglia/macrophage specific marker KiM1P, and the neuronal marker NeuN were performed on formalin-fixed brain specimens. Although mainly neurons of both PSN and controls expressed Fas receptor, expression was significantly increased (p = 0.001) in PSN cases in which predominantly degenerating cells with signs of early apoptosis showed Fas expression. In contrast, Fas ligand expression was found mainly on astrocytes and microglial cells. There was no significant difference between cases with and without PSN. We conclude that in the developing human brain, cells expressing the Fas receptor may be susceptible to undergoing apoptosis in response to hypoxic-ischemic injury.

Immunological detection of 4-hydroxynonenal protein adducts in developing pontine and Purkinje neurons and in karyorrhexis in pontosubicular neuronal necrosis.

Four-hydroxynonenal (HNE) has been proposed as an important marker of radical-induced lipid peroxidation. The principal objective of this study was to assess the occurrence of lipid peroxidation in normal perinatal brain and brains with one form of pontosubicular neuronal necrosis (PSN). Immunochemical studies using an antibody against HNE-modified protein were performed in controls aged from 20 weeks of gestation to 64 years, and patients with PSN. Immunohistochemical study showed developmental and aging changes of positive staining in Purkinje cells and pontine neurons (27 weeks-7 months, 50 and 64 years). In addition, karyorrhectic cells in pontine nuclei with PSN were positively stained. Immunoblotting revealed that a 75-kDa protein, which is speculated to be mitochondrial complex-1 protein, was the most intensely expressed among multiple immunoreactive proteins. Our results identified the presence of oxidative stress in the perinatal neuron, and this oxidative stress may contribute to some forms of karyorrhectic death.

Caspase-3 activation and caspase-like proteolytic activity in human perinatal hypoxic-ischemic brain injury.

Human perinatal hypoxic-ischemic brain injury is an important cause of death and morbidity. One relatively common pattern of perinatal injury involves selective neuronal death in the ventral gray matter of the pons and in the subiculum of the hippocampal formation, classically termed 'pontosubicular neuronal necrosis' (PSN). The vulnerable neurons undergo karyorrhectic condensation of their nuclear chromatin and exhibit in situ end labeling for DNA fragmentation, leading to the recent reclassification of cell death in PSN as apoptotic. Caspase activation plays a central role in apoptosis and caspase-3 appears to be an especially important effector enzyme in neuronal apoptosis. In this study we performed immunohistochemistry on brain sections from six postmortem cases of PSN using two polyclonal antisera; CM1, a specific marker of caspase-3 activation, and fractin, which specifically recognizes a neoepitope at a caspase cleavage site in actin, and is therefore a marker of caspase-like proteolytic activity. Numerous CM1- and fractin-immunolabeled neurons were seen in the nuclei pontis and subiculum in each case, and the great majority showed karyorrhectic morphology. The immunostaining involved the nuclei and cytoplasm of these cells and the proximal portions at least of their neuritic processes. Some neurons exhibited a more extensive pattern of dendritic fractin labeling. Frequent CM1- and fractin-immunoreactive axonal segments were also seen. The identification of caspase-3 activation and caspase-like proteolytic activity in PSN cases in this study suggests that caspase inhibitors may potentially have a therapeutic neuroprotective role in human perinatal hypoxic-ischemic brain injury.

Expression of cell death-associated proteins in neuronal apoptosis associated with pontosubicular neuron necrosis.

Expression of apoptosis-associated proteins p53, bcl-2, bax, and caspase-3/CPP32, activation of caspase-3, and modification of proteins via poly(ADP-ribosyl)ation was studied in pontosubicular neuron necrosis (PSN), a form of perinatal brain damage revealing the morphological hallmarks of neuronal apoptosis. Immunoreactivity for p53 was completely absent. The majority of cells stained with the bax and procaspase-3 antibodies did not show morphological signs of apoptosis. In contrast, an antibody against activated caspase-3 almost exclusively stained cells with apoptotic morphology. Poly(ADP-ribosyl)ated proteins were only rarely detected in cells with apoptotic morphology. The expression patterns of bax, procaspase-3, bcl-2, and p53 in PSN were similar to that found in age-matched control brains. However, activated caspase-3 and poly-ADP-ribosylated proteins were exclusively found in apoptotic cells. These data indicate that detection of active caspase-3 is a reliable marker for apoptosis in formalin-fixed human tissue, and that neuronal apoptosis in pontosubicular neuron necrosis is accompanied by a pronounced activation of caspase-3.

Roles of glutamate transporter and receptors, poly (ADPribose) polymerase, and transforming growth factor-beta1 in pontosubicular neuron necrosis.

The expression of neuron-type glutamate transporters (EAAC-1), AMPA glutamate receptor subunits (GluR1 and GluR2/3), polyadenosine (5'diphosphate-ribose) polymerase (PARP), and transforming growth factor-beta1 was investigated in 20 cases of neonatal pontosubicular neuron necrosis and 12 gestational-age matched controls. Developmental immunoreactivities of EAAC-1, GluR1, and GluR2/3 appeared in the neurons of the pontine nuclei at 29 to 30 weeks' gestation in controls, and then gradually increased with age. However, these activities were decreased in the pontine nucleus of patients with pontosubicular neuron necrosis. Decreases in these immunoreactivities might indicate early degeneration of neurons. Although PARP and transforming growth factor-beta1 immunoreactivity was insignificant or very weak in the pontine nuclei at any age in controls, PARP was markedly expressed in karyorrhectic neurons of the pontine nucleus in patients with pontosubicular neuron necrosis. Transforming growth factor-beta1 immunoreactivity was observed in nonkaryorrhectic neurons of the pontine nuclei. PARP could contribute to the pathogenesis of pontosubicular neuron necrosis more than EAAC-1 or GluR1 or GluR2/3. Transforming growth factor-beta1 could play a role in the protection and repair of damaged neurons.

Functional Change of NMDA Receptors Related to Enhancement of Susceptibility to Neurotoxicity in the Developing Pontine Nucleus

The developing neurons have been reported to be extremely susceptible to toxicity of NMDA during a restricted developmental period. Pontosubicular neuronal necrosis is a typical type of perinatal human brain lesion and often coexists with other forms of cerebral hypoxic and ischemic injuries. To determine whether functional changes of NMDA receptors related to the susceptibility to NMDA toxicity are involved in developing neurons in the pontine nucleus, we have examined the lesion produced by in vivo direct injection of NMDA into the pontine nucleus of rats at postnatal days 1-30, recorded NMDA-induced whole-cell currents from neurons in the pontine nucleus in the developing rat brainstem slices, and performed in situ hybridization for NMDA receptor subunit mRNAs in the pontine nucleus. The susceptibility to NMDA neurotoxicity peaked near postnatal day 15, and the NMDA-induced currents showed prominent reduction of the voltage-dependent block by Mg2+ near postnatal day 15. The pontine nucleus near postnatal day 15 showed distinct expression of the NMDA receptor subunit NR2C mRNA. These results suggest that the susceptibility to NMDA neurotoxicity that is enhanced in the rat pontine nucleus near postnatal day 15 is mediated by the NMDA receptor channels that are relatively insensitive to Mg2+ and that the reduction in the sensitivity of NMDA receptors to Mg2+ correlates with the expression of the NR2C. We present the possibility that functional changes in the NMDA receptor channels play a crucial role in the occurrence of developmentally specific neuronal injury.

Alzheimer disease: DNA fragmentation indicates increased neuronal vulnerability, but not apoptosis.

Although nerve cell loss is prominent in certain brain regions in Alzheimer disease (AD), it is currently unresolved how these cells die. Recent studies unanimously agree that there are more neurons displaying DNA fragmentation in AD compared with normal controls. However, controversy remains as to whether cell death is mediated by apoptosis or necrosis. We addressed this question by comparing AD lesions with those from cases with pontosubicular neuron necrosis (PSNN), a human pathological condition with unequivocal neuronal apoptosis, with regard to cell and nuclear morphology, immunohistochemistry, and in situ tailing. Immunohistochemistry was performed for an array of proteins with presumptive roles in the apoptotic process or the protection thereof, i.e. a recently described apoptosis-specific protein (ASP), the transcription factor c-Jun, Bcl-2, and various stress proteins: alpha B-Crystallin, heat shock protein (HSP) 27, HSP 65, HSP 70, HSP 90, and ubiquitin. Apoptotic neurons in PSNN displayed chromatin condensation, nuclear fragmentation, and cytoplasmic condensation. They were labeled with the in situ tailing technique and stained for the ASP. Despite the large numbers of cells with DNA fragmentation identified in the hippocampus of AD brains, only exceptional cells displayed the morphological characteristics of apoptosis or labeled for the ASP. We suggest that the increased rate of neuronal DNA fragmentation in AD patients indicates a higher susceptibility of the cells to metabolic disturbances compared with normal controls. The large number of cells with DNA fragmentation most likely reflects metabolic disturbances in the premortem period, and cell destruction is mediated through necrosis rather than apoptosis.

Cu, Zn-superoxide dismutase reaction in neonatal pontosubicular neuron necrosis

The immunohistochemical localization and changes in copper/zinc superoxide dismutase (Cu, Zn-SOD) were examined in 14 neonates with pontosubicular neuron necrosis (PSN), as compared with those in 15 controls in which the cytoplasm of neurons and glial cells showed SOD immunoreactivity. In the temporal lobes and hippocampus with PSN, Cu, Zn-SOD reactivity was negative in neurons at 0 and 1 days after birth, but was positive after 5 days of age in 8 of 10 cases. In the pons and cerebellum, SOD-positive neurons appeared soon after birth, but eosinophilic or karyorrhectic neurons were SOD negative. On the other hand, glial cells were positive after birth in all cases of PSN, and their reactivity was increased in the cases of reactive astrogliosis. Early loss of the scavenging system directed at free radicals may lead to neuronal damage, and the induction of Cu, Zn-SOD may act as a defense mechanism against damage of neurons in neonates with PSN. Therefore, oxygen-derived free radicals may be one of the pathogenetic factors of PSN with characteristics of apoptosis in neonates.

Transient expression of apolipoprotein-E in neonates with pontosubicular neuron necrosis.

An apolipoprotein-E (Apo-E) immunohistochemical study was performed on neonates with pontosubicular neuron necrosis (PSN), aged 38-42 weeks of gestation, and compared to findings for age-matched neonates without PSN. Apo-E was expressed in neurons in both the pontine nuclei and pyramidal layer of the hippocampus, as well as astrocytes of only the PSN cases. The immunoreactive neurons did not exhibit karyorrhexis and were found in neonates by the age of 6 days. Apo-E may be produced by astrocytes and taken up by neurons on membrane remodeling during early responses to cerebral hypoxic or ischemic insult in PSN neonates.

Evidence for neuronal apoptosis in pontosubicular neuron necrosis

Pontosubicular neuron necrosis (PSN) is characterized by acute neuronal death in the subiculum and the pons occurring in a circumscribed perinatal period. The morphological changes in PSN are quite similar to those described during apoptosis, a form of programmed cell death. Morphological re-evaluation of the lesions by light and electron microscopy revealed the typical changes of apoptosis with condensed basophilic nuclei and the formation of apoptotic bodies. By analysing DNA fragmentation in situ with a recently established technique, we were able to show that neuronal death in PSN is apoptotic. The demonstration of DNA fragmentation by the in situ tailing technique was reliable in the human autopsy material used in this study and was only slightly affected by autolysis or formalin fixation. The subiculum and the pons are shown to be susceptible to apoptosis at different times during development. PSN thus provides a model in which the process of nerve cell apoptosis in the developing human central nervous system can be studied.

The prenatal age critical for the development of the pontosubicular necrosis.

Pontosubicular neuronal necrosis is characterized by neuronal karyorrhexis, showing a peculiar distribution. In infants delivered at more than 29 gestational weeks (GW), neuronal karyorrhexis is restricted to the pons and subiculum, while in very premature infants (delivered at less than 28 GW), neurons in other brain regions, such as the inferior olivary nucleus, cerebellum, basal ganglia, thalamus and cerebral cortex, are also involved. Thus, karyorrhexis is more widely distributed in the more immature brain, implicating neuronal maturation as one of the pathogenetic factors relevant to this type of neuronal cell death.

Ferritin immunohistochemical study on pontine nuclei from infants with pontosubicular neuron necrosis

Immunohistochemical ferritin staining was performed on pontine nuclei of the brains of 17 infants with pontosubicular neuron necrosis (PSN), aged 23 to 42 weeks of gestation. Ferritin-positive cells were increased in cases of karyorrhexis with spongy changes and gliosis, but not in those of selective karyorrhexis. Ferritin-positive cells were more increased in the cases with extensive karyorrhectic neurons. Iron may be released to the damaged pontine tissue as a catalyst and microglia may play an important role in the repair of the tissue.