NDRG4, an early detection marker for colorectal cancer, is specifically expressed in enteric neurons.

BACKGROUND: Promoter methylation of N-myc Downstream-Regulated Gene 4 (NDRG4) in fecal DNA is an established early detection marker for colorectal cancer (CRC). Despite its connection to CRC, NDRG4 is predominantly studied in brain and heart, with little to no knowledge about its expression or role in other organs. In this study, we aimed to determine the whole-body expression of NDRG4, with a focus on the intestinal tract. METHODS: We investigated NDRG4 expression throughout the body by immunohistochemistry, Western Blotting and in situ mRNA hybridization using tissues from NDRG4 wild-type, heterozygous and knockout mice and humans. In addition, we explored cell-specific expression of NDRG4 in murine whole-mount gut preparations using immunofluorescence and confocal microscopy. KEY RESULTS: NDRG4 is specifically expressed within nervous system structures throughout the body. In the intestinal tract of both mouse and man, NDRG4 immunoreactivity was restricted to the enteric nervous system (ENS), where it labeled cell bodies of the myenteric and submucosal plexuses and interconnecting nerve fibers. More precisely, NDRG4 expression was limited to neurons, as NDRG4 always co-localized with HuC/D (pan-neuronal marker) but never with GFAP (an enteric glial cell marker). Furthermore, NDRG4 was expressed in various neuropeptide Y positive neurons, but was only found in a minority (~10%) of neurons expressing neuronal nitric oxide synthase. CONCLUSIONS AND INFERENCES: NDRG4 is exclusively expressed by central, peripheral and enteric neurons/nerves, suggesting a neuronal-specific role of this protein. Our findings raise the question whether NDRG4, via the ENS, an understudied component of the tumor microenvironment, supports CRC development and/or progression.

Fetal asphyxia leads to the loss of striatal presynaptic boutons in adult rats.

Fetal asphyxic insults in the brain are known to be associated with developmental and neurological problems like neuromotor disorders and cognitive deficits. Little is known, however, about the long-term consequences of fetal asphyxia contributing to the development of different neurological diseases common in the adult or the aging brain. For that reason the present study aimed to investigate the long-term effects of fetal asphyxia on synaptic organization within the adult rat brain. Fetal asphyxia was induced at embryonic day 17 by 75-min clamping of the uterine and ovarian arteries. Presynaptic bouton densities and numbers were analyzed in the striatum and prefrontal cortex at the age of 19 months. A substantial decrease in presynaptic bouton density and number was observed in the striatum of fetal asphyxia rats compared to control rats, while an increase was found in the fifth layer of the prefrontal cortex. These results suggest that fetal asphyxia can have long-lasting effects on synaptic organization that might contribute to a developmental etiology of different neurological disorders and aging.

A combined behavioral and morphological study on the effects of fetal asphyxia on the nigrostriatal dopaminergic system in adult rats.

Fetal asphyxic insults in the brain are known to be associated with developmental neurological problems like neuromotor disorders. However, little is known about the long-term consequences of fetal asphyxia (FA). For that reason, the present study investigated the long-term effects of FA on motor behavior and dopaminergic circuitry. FA was induced at embryonic day 17 by 75-minute clamping of the uterine circulation. SHAM animals underwent the same procedure except for the clamping. This was followed by full-term vaginal delivery of animals in all groups (FA, SHAM and untreated controls). At 6 months, basal and amphetamine-induced locomotor activity was measured during open field testing. Brain sections were stained for tyrosine hydroxylase (TH) and glial fibrillary acidic protein (GFAP). TH-positive cells and GFAP-positive cells in substantia nigra pars compacta (SN(C)) and striatum were counted using design-based stereology. Moreover, TH-immunoreactivity in the striatum was assessed by grey value measurements. Behavioral analysis demonstrated that SHAM and FA showed less basal and amphetamine-induced activity than controls. Histochemically, FA decreased the number of TH-positive neurons in the SN(C) and lowered TH-positive in the striatum. Furthermore, more GFAP-positive cells were found in the SN(C) and striatum in FA than in either control and SHAM groups. Additionally, FA animals showed ventriculomegaly associated with smaller white matter as well as grey matter volumes. The data show that FA was associated with deficits in both dopamine-related motor behavior and biochemistry. These alterations were associated with nigrostriatal astrogliosis. The present study demonstrates the sensitivity of the dopaminergic system towards FA.

Prenatal restraint stress and long-term affective consequences.

Chronic or repeated stress during critical periods of human fetal brain development has been associated with various learning, behavioral and/or mood disorders in later life. In this investigation, pregnant Fischer 344 rats was individually restrained three times a day for 45 min during the last week of gestation in transparent plastic cylinders while at the same time being exposed to bright light. Control pregnant females were left undisturbed in their home cages. Anxiety and depressive-like behavior was measured in the offspring at an age of 6 months using the open field test, the home cage emergence test and the forced swim test. Prenatally stressed rats spent more time in the corners and less time along the walls of an open field, while no difference in total distance moved was observed. In addition, prenatally stressed rats took more time to leave their home cage in the home cage emergence test. On the other hand, no differences in immobility were observed in the forced swim test. Moreover, prenatally stressed rats showed lower stress-induced plasma corticosterone levels compared with control rats. Prenatal stress (PS) had no effect on the number of 5-bromo-2-deoxyuridine-positive cells - used as a measure for cell proliferation - in the dentate gyrus of these rats. These data further support the idea that PS may perturb normal anxiety-related development. However, the present data also suggest that an adaptive or protective effect of PS should not be ignored. Genetic factors are likely to play a role in this respect.

Apoptosis in the rat spinal cord during postnatal development; the effect of perinatal asphyxia on programmed cell death.

The aim of our study was to investigate the effect of perinatal asphyxia on developmental apoptosis in the cervical and lumbar spinal cord in the neonatal rat. Perinatal asphyxia was induced by keeping pups at term in utero in a water bath at 37 degrees C for 20 min, followed by resuscitation. Effects of this treatment on developmental apoptosis were studied on postnatal days 2, 5 and 8 using terminal deoxynucleotidyl transferase (TdT)-dUTP-biotin nick end labelling (TUNEL) and caspase-3 staining. TUNEL positive cells were identified using double immunostaining. On postnatal day 2 an increase of 215% in TUNEL positive cells was detected (P=0.005) in laminae IV-VII of the lumbar spinal cord of rats which underwent perinatal asphyxia compared to controls. An increase of 55% compared to controls (P=0.03) was seen in laminae I-III of the lumbar spinal cord at postnatal day 8. TUNEL positive cells could be partly identified as microglia cells (ED1 positive) and oligodendrocytes (O4 positive). The effect of perinatal asphyxia on programmed cell death in the neonatal rat spinal cord was mainly observed in the intermediate zone and dorsal horn of the lumbar spinal cord. We conclude that perinatal asphyxia has a pronounced effect on the survival of cells in a specific region of the spinal cord and thus may have a profound effect on the development of motor networks.

Baclofen inhibits ANP-mediated cyclic GMP synthesis in the rat cervical spinal cord.

The neurotransmitter gamma-aminobuteric acid (GABA) is believed to have a controlling action on spinal locomotor networks. In spasticity, spinal locomotor networks are thought to play a role. A well known drug in the treatment of spasticity is the GABA(B) agonist Baclofen. We report an inhibitory effect of Baclofen on the ANP-mediated cGMP synthesis in the superficial dorsal horn (laminae I-III) of the rat cervical spinal cord. This inhibitory effect of Baclofen could not be detected after incubation with the NO donor SNP. The clinical effect of Baclofen on the reduction of spasticity might be explained by an enhancement of GABAergic inhibition of ANP mediated cGMP concentration in the spinal cord dorsal horn, thus reducing afferent input.

Developmental apoptosis in the spinal cord white matter in neonatal rats.

We investigated developmental apoptosis in the white matter of the cervical spinal cord at postnatal days 2, 5, and 8. Apoptotic cells were labeled using TUNEL and caspase-3 immunostaining. Apoptotic cells were diffusely distributed throughout the white matter of the spinal cord. The total amount of apoptotic cells in the cervical spinal cord white matter was related to postnatal age, with the lowest at P2 (mean 7.9, SD 5.6) and the highest at P8 (mean 109, SD 21.4). Using double immunostaining for ED-1 and O4, apoptotic cells were identified as microglia and oligodendrocytes.