Using the Optical Fractionator to Estimate Total Cell Numbers in the Normal and Abnormal Developing Human Forebrain.

Human fetal brain development is a complex process which is vulnerable to disruption at many stages. Although histogenesis is well-documented, only a few studies have quantified cell numbers across normal human fetal brain growth. Due to the present lack of normative data it is difficult to gauge abnormal development. Furthermore, many studies of brain cell numbers have employed biased counting methods, whereas innovations in stereology during the past 20-30 years enable reliable and efficient estimates of cell numbers. However, estimates of cell volumes and densities in fetal brain samples are unreliable due to unpredictable shrinking artifacts, and the fragility of the fetal brain requires particular care in handling and processing. The optical fractionator design offers a direct and robust estimate of total cell numbers in the fetal brain with a minimum of handling of the tissue. Bearing this in mind, we have used the optical fractionator to quantify the growth of total cell numbers as a function of fetal age. We discovered a two-phased development in total cell numbers in the human fetal forebrain consisting of an initial steep rise in total cell numbers between 13 and 20 weeks of gestation, followed by a slower linear phase extending from mid-gestation to 40 weeks of gestation. Furthermore, we have demonstrated a reduced total cell number in the forebrain in fetuses with Down syndome at midgestation and in intrauterine growth-restricted fetuses during the third trimester.

Comparison of analytical methods of brain [18F]FDG-PET after severe traumatic brain injury.

BACKGROUND: Loss of consciousness has been shown to reduce cerebral metabolic rates of glucose (CMRglc) measured by brain [18F]FDG-PET. Measurements of regional metabolic patterns by normalization to global cerebral metabolism or cerebellum may underestimate widespread reductions. NEW METHOD: The aim of this study was to compare quantification methods of whole brain glucose metabolism, including whole brain [18F]FDG uptake normalized to uptake in cerebellum, normalized to injected activity, normalized to plasma tracer concentration, and two methods for estimating CMRglc. Six patients suffering from severe traumatic brain injury (TBI) and ten healthy controls (HC) underwent a 10min static [18F]FDG-PET scan and venous blood sampling. RESULTS: Except from normalizing to cerebellum, all quantification methods found significant lower level of whole brain glucose metabolism of 25-33% in TBI patients compared to HC. In accordance these measurements correlated to level of consciousness. COMPARISON WITH EXISTING METHODS: Our study demonstrates that the analysis method of the [18F]FDG PET data has a substantial impact on the estimated whole brain cerebral glucose metabolism in patients with severe TBI. Importantly, the SUVR method which is often used in a clinical setting was not able to distinguish patients with severe TBI from HC at the whole-brain level. CONCLUSION: We recommend supplementing a static [18F]FDG scan with a single venous blood sample in future studies of patients with severe TBI or reduced level of consciousness. This can be used for simple semi-quantitative uptake values by normalizing brain activity uptake to plasma tracer concentration, or quantitative estimates of CMRglc.

Spatiotemporal distribution of PAX6 and MEIS2 expression and total cell numbers in the ganglionic eminence in the early developing human forebrain.

The development of the human neocortex is a complex and highly regulated process involving a time-related expression of many transcription factors including the homeobox genes Pax6 and Meis2. During early development, Pax6 is expressed in nuclei of radial glia cells in the neocortical proliferative zones and controls the differentiation and neurogenetic fate of these cells in the dorsal telencephalon in rodents. Animal studies on the Meis2 gene have revealed expression in the developing telencephalon and Meis2 is known to regulate the expression of Pax6 in the eye and pancreas. Because of this functional relation between Pax6 and Meis2, we studied the spatial and temporal expression of PAX6, and MEIS2 using a developmental series of human fetal brains at 7-19 postconceptional weeks with emphasis on the forebrain to investigate whether the two genes are expressed in the same regions and zones in the same time window. We demonstrate by in situ hybridization and immunohistochemistry that the two homeobox genes are expressed during early fetal brain development in humans. PAX6 mRNA and protein were located in the proliferative zones of the neocortex and in single cells in the cortical preplate at 7 fetal weeks and in the developing cortical plate from 8 or 9 to 19 fetal weeks. The expression of PAX6 expanded into the ganglionic eminence just prior to the stage at which a stereological estimation showed an exponential rise in total cell number in this area. The MEIS2 gene was also present in the proliferative zones of the human fetal neocortex and a higher expression of MEIS2 than PAX6 was observed in these areas at 9 fetal weeks. Further, MEIS2 was expressed at a very high level in the developing ganglionic eminence and at a more moderate level in the cortical plate.

Expression of the homeobox genes OTX2 and OTX1 in the early developing human brain.

In rodents, the Otx2 gene is expressed in the diencephalon, mesencephalon, and cerebellum and is crucial for the development of these brain regions. Together with Otx1, Otx2 is known to cooperate with other genes to develop the caudal forebrain and, further, Otx1 is also involved in differentiation of young neurons of the deeper cortical layers. We have studied the spatial and temporal expression of the two homeobox genes OTX2 and OTX1 in human fetal brains from 7 to 14 weeks postconception by in situ hybridization and immunohistochemistry. OTX2 was expressed in the diencephalon, mesencephalon, and choroid plexus, with a minor expression in the basal telencephalon. The expression of OTX2 in the hippocampal anlage was strong, with no expression in the adjacent neocortex. Contrarily, the OTX1 expression was predominantly located in the proliferative zones of the neocortex. At later stages, the OTX2 protein was found in the subcommissural organ, pineal gland, and cerebellum. The early expression of OTX2 and OTX1 in proliferative cell layers of the human fetal brain supports the concept that these homeobox genes are important in neuronal cell development and differentiation: OTX1 primarily in the neocortex, and OTX2 in the archicortex, diencephalon, rostral brain stem, and cerebellum.

Expression of the homeobox genes PAX6, OTX2, and OTX1 in the early human fetal retina.

We studied the spatial and temporal expression of the homeobox genes PAX6, OTX2, and OTX1 using a developmental series of human fetal eyes aged from 6 to 10 weeks post-conception. Previous animal studies have shown that PAX6 may regulate progenitor cell proliferation, timing of differentiation and neural retina cell fate determination. OTX2 may play a role in development of the retinal pigment epithelium and photoreceptor differentiation, whereas OTX1 may be important in ciliary body development. In this study, we demonstrate the presence of the mRNAs and proteins for these genes within human fetal retinas of different stages. By in situ hybridization and immunohistochemistry, we show that PAX6 was primarily localized to nuclei of the neural retina, OTX2 was localized to the nuclei of retinal pigment epithelium and OTX1 expression was confined to anterior retina. Expression peaks for PAX6 occur at days 51-60 and for OTX2 occur around fetal days 48-54. We conclude that the human expression patterns correspond spatially with the patterns observed in the rat retina. Further, the expressions occur during similar fetal stages as described in the developing rat retina.