Damaso Crespo

Changes in the numbers of optic nerve fibers during late prenatal and postnatal development in the albino rat

We have estimated from electron micrographs the numbers of axons in the optic nerves of a series of albino rats at 8 different ages ranging from embryonic day (E)18 through postnatal day 28. The number of axons was found to reach a maximum of about 325,000 (324,790 +/- 38,589) on E20, and to decline to about 275,000 (273,744 +/- 20,973) by the day of birth. By the middle of the second postnatal week, the number was further reduced to a stable figure of just over 100,000 (105,809 +/- 7,610). This represents a loss of two-thirds of the axons from the peak value at E20, and suggests that there is a comparable degree of cell death among the retinal ganglion cells. The reduction in the number of optic nerve fibers is not affected significantly by the removal of the opposite eye at birth.

Hippocampal volume and neuronal number in Ts65Dn mice: a murine model of down syndrome

Ts65Dn mouse displays a partial triplication of chromosome 16 and is adopted as a model for Down syndrome (DS). It is known that Ts65Dn mice present memory deficiencies. In order to gain insight into the cause of these deficiencies, we studied the possibility of changes in volumes and neuronal numbers in different regions of the hippocampus (dentate gyrus, CA3, CA2 and CA1) in trisomic mice as compared to control littermates using stereological methods. The mean hippocampal volumes of Ts65Dn mice did not show significant differences as compared to controls, except in CA2 where there was a barely significant decrease. However, mean neuron number was significantly lower in Ts65Dn mice than in controls in dentate gyrus (43.7 x 10(4), CV 21%, n = 5, vs. 30.4 x 10(4), CV 18.1%, n = 4) and higher in CA3 (23.1 x 10(4), CV 18.9% vs. 33.3 x 10(4), CV 14.9%). These quantitative changes may account for the memory deficiencies observed in Ts65Dn mice.

How does chondroitinase promote functional recovery in the damaged CNS

A number of recent studies have established that the bacterial enzyme chondroitinase ABC promotes functional recovery in the injured CNS. The issue of how it works is rarely addressed, however. The effects of the enzyme are presumed to be due to the degradation of inhibitory chondroitin sulphate GAG chains. Here we review what is known about the composition, structure and distribution of the extracellular matrix in the CNS, and how it changes in response to injury. We summarize the data pertaining to the ability of chondroitinase to promote functional recovery, both in the context of axon regeneration and the reactivation of plasticity. We also present preliminary data on the persistence of the effects of the enzyme in vivo, and its hyaluronan-degrading activity in CNS homogenates in vitro. We then consider precisely how the enzyme might influence functional recovery in the CNS. The ability of chondroitinase to degrade hyaluronan is likely to result in greater matrix disruption than the degradation of chondroitin sulphate alone.

Chondroitinase ABC has a long-lasting effect on chondroitin sulphate glycosaminoglycan content in the injured rat brain.

Chondroitin sulphate proteoglycans (CSPGs) are axon growth inhibitory molecules present in the glial scar that play a part in regeneration failure after damage to the CNS and which restrict CNS plasticity. Removal of chondroitin sulphate glycosaminoglycan (GAG) chains with chondroitinase‐ABC (chABC) in models of CNS injury promotes both axon regeneration and plasticity. We have analysed the immediate and long‐term effects of a single injection of chABC on CSPGs, GAGs and axon regeneration. We made unilateral nigrostriatal lesions in adult rats accompanied by an adjacent infusion of either chABC or a bacterial‐derived control enzyme (penicillinase). Within 24 h of chABC treatment there was digestion of GAGs, including hyaluronan, and a reduction in neurocan in an area extending 1.5 mm around the injection site. Around 50% of GAG is inaccessible to chABC digestion, even in tissue digested in vitro, which probably represents intracellular stores. In control penicillinase treated animals, total GAGs recovered from the lesioned brains were up‐regulated by 4‐fold 7 days after injury and gradually decreased to normal at 28 days post‐lesion. In chondroitinase‐treated animals, the total GAG remained at low level throughout the 28‐day experimental period. This suggests the persistence of active chABC for at least 10 days after injection which is able to digest CSPGs released from cells during this time. This was confirmed by immunological detection of enzyme for 10 days and by retrieval of active enzyme from the brain at 10 days after injection. Our results suggest that a single injection of chABC can produce an environment conducive to CNS repair for over 10 days.

Transient c-fos expression accompanies naturally occurring cell death in the developing interhemispheric cortex of the rat

We have searched for the possible correlation of naturally occurring cell death with spontaneously enhanced c-fos expression in the developing cerebral cortex of normal Wistar albino rats. During the late prenatal and early postnatal period, cells with irregular contours and intracytoplasmic electron-dense granules (granule-containing cells) were apparent in the interhemispheric cortex, including the anterior cingulate and the retrosplenial cortices. These cells were loosely packed within the cortical layers derived from the cortical plate. Having excluded the possibility that these cells could be phagocytes by immunocytochemical experiments, we propose that they are cells in different phases of a process of autophagic degeneration and death. Images of extreme nuclear pyknosis were also apparent in identical locations. Cells showing immunoreactivity for c-Fos protein appeared in the same cortical areas. The immunoreactive cells were very abundant in the retrosplenial cortex, but were also present in the anterior cingulate cortex. These cells showed markedly irregular contours and large, densely immunoreactive intracytoplasmic inclusions; these images were similar to those of granule-containing cells revealed by conventional stains. The immunoreactivity for c-Fos protein was ephemeral, occurring exclusively during embryonic days 20 and 21, but granule-containing cells were observed for a longer period. The present results provide evidence, albeit indirect, that c-fos expression may occur in certain neural cells at the onset of a process of death by autophagia, and suggest a possible involvement of the proto-oncogene c-fos in certain forms of naturally occurring neuronal death.

Effects of melatonin on the proliferation and differentiation of human neuroblastoma cells in culture

Since melatonin has direct inhibitory effects on some tumor cells in vitro, the aim of the present work was to study whether the growth and structural characteristics of the human neuroblastoma cell line SK-N-SH in vitro are influenced by this indoleamine. Concentrations of melatonin of 10(-9) and 10(-11) M significantly inhibited (P < 0.05) cell proliferation. Subphysiological (10(-13) M) or supraphysiological (10(-7) and 10(-5) M) concentrations of melatonin lacked this effect. After 8 days of exposure to melatonin (10(-9) M), cells showed significantly smaller cell and nuclear sizes than control cells. Melatonin-treated cells presented greater neurite outgrowth than control cells. These results support the hypothesis that melatonin, at physiological concentrations, exerts a direct antiproliferative effect on SK-N-SH cells, promoting the differentiation of neuroblastoma cells.

The effect of intraocular tetrodotoxin on the postnatal reduction in the numbers of optic nerve axons in the rat

We have examined the effects of blocking retinal ganglion cell activity with the sodium channel blocker tetrodotoxin (TTX) on the postnatal reduction in the number of optic nerve axons (and, by interference, the degree of ganglion cell loss in the retina). TTX was injected every other day into the left eyes of a series of albino rats beginning on the day of birth, continuing through the 3rd, 7th, 12th or 14th days when the animals were killed and the optic nerves from both eyes were prepared for electron microscopy. The numbers of axons in the TTX treated and untreated optic nerves from the opposite side were determined from electron micrographs, and compared to the number seen in normal rats at the same ages. Both the magnitude and the time course of the reduction in the number of axons in the TTX-treated and untreated nerves were found to be similar to those seen in normal animals. However, there was a slight reduction in the loss of optic axons in the untreated nerves on the side opposite the TTX injections; this attenuation in axon loss could be mimicked by large systemic injections of TTX, and is probably attributable to a general systemic effect following repeated intraocular injections. These findings indicate that blocking ganglion cell activity with intraocular injections of TTX has little effect on the normal rate of axon loss from the optic nerve and on the numbers of ganglion cells that die during the first two weeks of postnatal life.

Nucleoli numbers and neuronal growth in supraoptic nucleus neurons during postnatal development in the rat.

We present a quantitative study of the variations in the number of nucleoli in supraoptic nucleus neurons during the postnatal period, as well as a morphometric and stereological analysis of the nuclear and cytoplasmic volume changes of these maturing neurons. The mean number of nucleoli per cell was 1.59 +/- 0.28 (mean +/- S.D.) at P1; it then began to decrease until P14 (1.32 +/- 0.67) at which age the adult pattern in the number of nucleoli was attained. The mean nuclear volume increased steadily from 214.56 +/- 6.48 microns 3 (mean +/- S.E.) at P1 to 326.1 +/- 10.93 microns 3 at P14 where it remained constant. The average cytoplasmic volume underwent a remarkable increase during postnatal period from 256.38 +/- 12.66 microns 3 at P1 to 3791.18 +/- 204.88 microns 3 at P90. It is noteworthy that the stabilization of the number of nucleoli coincides with the termination of the nuclear growth phase of supraoptic neurons. We suggest that these nuclear and nucleolar changes reflect the attainment of the fully-differentiated state of the protein synthesis machinery in these neurosecretory neurons.

Cholinergic, serotonergic and catecholaminergic neurons are not affected in Ts65Dn mice.

TS65DN mice were developed as a model of Down syndrome (DS); they are trisomic for the distal segment of chromosome 16 (MMU16), which contains genes syntenic with some of the genes located on the critical region of human chromosome 21 (HSA21). Since behavioral and neurochemical disturbances have been observed in this animal model, it seemed interesting to perform an immunohistochemical characterization of the main cholinergic, catecholaminergic and serotonergic nuclei. However, when the brains of Ts65Dn mice were compared with those of control littermates, no differences were found either in the morphology of the neurons of the three systems or in the number of immunoreactive cells. The results indicate that these systems are not affected by the triplication of some of the genes present on chromosome 16.

Differences between the circadian system of two strains of senescence-accelerated mice (SAM).

The aim of this study was to look for morphological or functional differences between the circadian system of two substrains of Senescence Accelerated Mice (SAM): senescence-prone (SAMP8; average median survival time = 10.0 months) and senescence-resistant (SAMR1; average median survival time = 18.9 months). Neither the general structure nor the quantitative analysis of the number of VP-positive neurons in matched sections revealed differences between SAMP8 and SAMR1 mice. Under LD 12/12 photoperiod, all animals showed the typical pattern of wheel running activity with maximum activity in the first half of darkness period. The period of the locomotor activity in free running as well as the time course for synchronization after a 6 h phase delay of LD cycle were similar in both strains of SAM mice. However, SAMP8 animals reentrained significantly earlier (3.4 +/- 0.4 days) than SAMR1 (6.3 +/- 0.9 days) after a 6 h phase advance of LD cycle. We conclude that the circadian system of SAMP8 mice presents some functional differences with that of SAMR1, which could help to explain their different rate of ageing.