calbindin-D28k Immunoreactivity Research Articles

ИММУНОГИСТОХИМИЧЕСКОЕ ИССЛЕДОВАНИЕ КАЛЬБИНДИНАВ РАЗВИВАЮЩЕМСЯ МОЗЖЕЧКЕ КРЫСЫ

Background. Calbindin is a calcium-binding protein that supports calcium homeostasis for the normal functioning of neurons. Purpose. To study the distribution of immunoreactivity of calbindin-D28K in the structures of the developing cerebellum of the rat.Material and methods. The study was performed on 16 outbred white rats of different age groups: 2-, 7-, 15-days (early postnatal period), 45-days (puberty period). The cerebellum samples were taken and fixed in zinc-ethanol-formaldehyde for immunohistochemistry. Calbindin-D28K immunoreactivity was determined on paraffin sections using primary polyclonal rabbit antibodies.Results. In the cerebellar cortex, calbindin immunoreactivity was detected on the 2nd day after development of Purkinje cells (PC) in their perikaryons, and by the 15th day in their dendrites and it did not change by the 45th day. In all terms of the study in PC, it was detected not only in the cytoplasm, but also in their nucleus. In the granular layer, calbindin immunoreactivity decreased in rats in postnatal ontogenesis, however, in adult rats, some neurons were moderately immunopositive. Among them, from the 15th day after birth, the calbindin-immunoreactive afferent nerve fibers running in the white matter were detected. There were no significant differences in the distribution of calbindin between the lobes of the cerebellum of different phylogenetic age. Conclusions. Considering that the expression of сalbindin-D28k is detected throughout the entire period of development of Purkinje cells, as well as its physiological role in maintaining the function and homeostasis of calcium in them, it can be concluded that сalbindin-D28k is a valuable marker for the morphofunctional characteristics of PC in the developing and adult cerebellum of rats in normal and pathological conditions.

Calbindin-D28K, parvalbumin, and calretinin in young and aged human locus coeruleus

Certain neuronal populations, including basal forebrain cholinergic neurons (BFCN) and noradrenergic neurons of the locus coeruleus (LC), are selectively vulnerable to pathology and loss early in the course of aging and Alzheimer's disease (AD). Human BFCN show substantial loss of the calcium-binding protein (CBP), calbindin-D28K (CB), during normal aging, which is associated with formation of neurofibrillary tangles and BFCN loss in AD. Here we determined if, similar to the BFCN, LC neurons contain CB or the other 2 ubiquitous CBPs parvalbumin and calretinin, and whether these proteins display an age-related loss from LC neurons. Immunostaining for CBP and tyrosine hydroxylase, a marker of catecholaminergic neurons, was used in sections from the LC of young and aged human brains. Parvalbumin and calretinin immunoreactivities were completely absent from human LC neurons. A subpopulation of LC neurons (~10%) contained CB immunoreactivity. Quantitative analysis revealed no age-related loss of CB from LC neurons. Thus, unlike the BFCN, age-related loss of CB does not figure prominently in the selective vulnerability of LC neurons to degeneration in AD.

A Potential Role for the Existence of Pericytes in the Neurovascular Unit of the Sexually Dimorphic Nucleus of the Rat Preoptic Area to Control Blood-Brain Barrier Function.

The present study aimed at determining pericytes, a missing component in the previously proposed living neurovascular unit (NVU) of the sexually dimorphic nucleus of the preoptic area (SDN-POA) in rats. Calbindin D28K-immunoreactivities (CB28-irs) were used to delineate the SDN-POA in which CD13-immunoreactivities (CD13-irs) or alpha-smooth muscle actinimmunoreactivities (αSMA-irs), two pericyte biomarkers serving the indexes of pericytes, were tagged using two adjacent brain sections (90-micron intervals). In addition, the nestinimmunoreactive (nestin-ir) cells in the SDN-POA were counted as pericytes referring to additional standards: location and nucleic and cellular morphology. Male SDN-POA volume (5.0±0.3x10-3 mm3) was significantly larger than the female (1.7±0.3x10-3 mm3). Within the SDN-POA, the CD13-irs were characterized as dots, densely packed and net-like in distribution, while the αSMAirs, excluding pipe-like or circular structures, appeared as short rod-like structures that were sparsely distributed. The immunoreactive counts of alpha-smooth muscle actin were 353±57/mm2 in males and 124±46/mm2 in females (p<0.05). On the other hand, densities of the dot-like CD13-irs were similar between males (4009±301/mm2) and females (4018±414/ mm2). There was no difference between the male and the female in the nestin-ir pericyte count in the SDN-POA. In conclusion, the present study adds new information concerning pericytes to the living NVU of the SDN-POA. There is a difference of sex in the count of the αSMA-irs in the living NVU of the SDN-POA. However, why such a difference exists warrants further investigations.

Muscle spindle reinnervation using transplanted embryonic dorsal root ganglion cells after peripheral nerve transection in rats

ObjectivesMuscle spindles are proprioceptive receptors in the skeletal muscle. Peripheral nerve injury results in a decreased number of muscle spindles and their morphologic deterioration. However, the muscle spindles recover when skeletal muscles are reinnervated with surgical procedures, such as nerve suture or nerve transfer. Morphological changes in muscle spindles by cell transplantation procedure have not been reported so far. Therefore, we hypothesized that transplantation of embryonic sensory neurons may improve sensory neurons in the skeletal muscle and reinnervate the muscle spindles.Materials and methodsWe collected sensory neurons from dorsal root ganglions of 14‐day‐old rat embryos and prepared a rat model of peripheral nerve injury by performing sciatic nerve transection and allowing for a period of one week before which we performed the cell transplantations. Six months later, the morphological changes of muscle spindles in the cell transplantation group were compared with the naïve control and surgical control groups.ResultsOur results demonstrated that transplantation of embryonic dorsal root ganglion cells induced regeneration of sensory nerve fibre and reinnervation of muscle spindles in the skeletal muscle. Moreover, calbindin D‐28k immunoreactivity in intrafusal muscle fibres was maintained for six months after denervation in the cell transplantation group, whereas it disappeared in the surgical control group.ConclusionsCell transplantation therapies could serve as selective targets to modulate mechanosensory function in the skeletal muscle.

The Immunoreactivity of Calbindin on Cerebellum Cortex and Hippocampus of Mice Brain after One Week Exposure to Radiofrequency Radiation

Background & Objectives:Radiofrequency radiation affects the calcium permeability and accordingly this induces pathophysiological changes in the brain and its subsequent output in the brain as tumor genesis, neural degeneration, and cognition or behavior changes. Hence, we investigated the effects of radiofrequency (RF) radiation on calcium binding proteins, reactive gliosis, and tumor genesis in mouse brain. Materials & Methods:Mice were exposed to radiofrequency radiation in the cage chamber with average specific absorption rate (SAR) of 1.6 W/kg and 4.0 W/Kg for one hour per day for five days. For histochemical studies, radiofrequency exposed brains were compared with sham control using calbindin D-28k antibody.Results:The immunoreactivity of calbindin D-28k, a marker for calcium homeostasis was found to increase in dose-response manner in hippocampus and cerebellar cortex.Conclusion:The short-term exposure to 835 MHz RF could induce disruption of calcium homeostasis and resulting neurobehavioral changes or brain tumor.

The Immunoreactivity of Calbindin on Cerebellum Cortex and Hippocampus of Mice Brain After One Week Exposure To Radiofrequency Radiation.

Background &amp; Objectives:Radiofrequency radiation affects the calcium permeability and accordingly this induces pathophysiological changes in the brain and its subsequent output in the brain as tumor genesis, neural degeneration, and cognition or behavior changes. Hence, we investigated the effects of radiofrequency (RF) radiation on calcium binding proteins, reactive gliosis, and tumor genesis in mouse brain. Materials &amp; Methods:Mice were exposed to radiofrequency radiation in the cage chamber with average specific absorption rate (SAR) of 1.6 W/kg and 4.0 W/Kg for one hour per day for five days. For histochemical studies, radiofrequency exposed brains were compared with sham control using calbindin D-28k antibody.Results:The immunoreactivity of calbindin D-28k, a marker for calcium homeostasis was found to increase in dose-response manner in hippocampus and cerebellar cortex.Conclusion:The short-term exposure to 835 MHz RF could induce disruption of calcium homeostasis and resulting neurobehavioral changes or brain tumor.

Immunoreactivities of calbindin‑D28k, calretinin and parvalbumin in the somatosensory cortex of rodents during normal aging.

Calbindin-D28k (CB), calretinin (CR) and parvalbumin (PV), which regulate cytosolic free Ca2+ concentrations in neurons, are chemically expressed in γ-aminobutyric acid (GABA)ergic neurons that regulate the degree of glutamatergic excitation and output of projection neurons. The present study investigated age-associated differences in CB, CR and PV immunoreactivities in the somatosensory cortex in three species (mice, rats and gerbils) of young (1 month), adult (6 months) and aged (24 months) rodents, using immunohistochemistry and western blotting. Abundant CB-immunoreactive neurons were distributed in layers II and III, and age-associated alterations in their number were different according to the species. CR-immunoreactive neurons were not abundant in all layers; however, the number of CR-immunoreactive neurons was the highest in all adult species. Many PV-immunoreactive neurons were identified in all layers, particularly in layers II and III, and they increased in all layers with age in all species. The present study demonstrated that the distribution pattern of CB-, CR- and PV-containing neurons in the somatosensory cortex were apparently altered in number with normal aging, and that CB and CR exhibited a tendency to decrease in aged rodents, whereas PV tended to increase with age. These results indicate that CB, CR and PV are markedly altered in the somatosensory cortex, and this change may be associated with normal aging. These findings may aid the elucidation of the mechanisms of aging and geriatric disease.

Neuroprotective effects of ischemic preconditioning on hippocampal CA1 pyramidal neurons through maintaining calbindin D28k immunoreactivity following subsequent transient cerebral ischemia.

Ischemic preconditioning elicited by a non-fatal brief occlusion of blood flow has been applied for an experimental therapeutic strategy against a subsequent fatal ischemic insult. In this study, we investigated the neuroprotective effects of ischemic preconditioning (2-minute transient cerebral ischemia) on calbindin D28k immunoreactivity in the gerbil hippocampal CA1 area following a subsequent fatal transient ischemic insult (5-minute transient cerebral ischemia). A large number of pyramidal neurons in the hippocampal CA1 area died 4 days after 5-minute transient cerebral ischemia. Ischemic preconditioning reduced the death of pyramidal neurons in the hippocampal CA1 area. Calbindin D28k immunoreactivity was greatly attenuated at 2 days after 5-minute transient cerebral ischemia and it was hardly detected at 5 days post-ischemia. Ischemic preconditioning maintained calbindin D28k immunoreactivity after transient cerebral ischemia. These findings suggest that ischemic preconditioning can attenuate transient cerebral ischemia-caused damage to the pyramidal neurons in the hippocampal CA1 area through maintaining calbindin D28k immunoreactivity.

The distribution of calbindin-D28k, parvalbumin, and calretinin immunoreactivity in the inferior colliculus of circling mouse.

The circling mice with tmie gene mutation are known as an animal deafness model, which showed hyperactive circling movement. Recently, the reinvestigation of circling mouse was performed to check the inner ear pathology as a main lesion of early hearing loss. In this trial, the inner ear organs were not so damaged to cause the hearing deficit of circling (cir/cir) mouse at 18 postnatal day (P18) though auditory brainstem response data indicated hearing loss of cir/cir mice at P18. Thus, another mechanism may be correlated with the early hearing loss of cir/cir mice at P18. Hearing loss in the early life can disrupt the ascending and descending information to inferior colliculus (IC) as integration site. There were many reports that hearing loss could result in the changes in Ca2+ concentration by either cochlear ablation or genetic defect. However, little was known to be reported about the correlation between the pathology of IC and Ca2+ changes in circling mice. Therefore, the present study investigated the distribution of calcium-binding proteins (CaBPs), calbindin-D28k, parvalbumin, and calretinin immunoreactivity (IR) in the IC to compare among wild-type (+/+), heterozygous (+/cir), and homozygous (cir/cir) mice by immunohistochemistry. The decreases of CaBPs IR in cir/cir were statistically significant in the neurons as well as neuropil of IC. Thus, this study proposed overall distributional alteration of CaBPs IR in the IC caused by early hearing defect and might be helpful to elucidate the pathology of central auditory disorder related with Ca2+ metabolism.

Detrimental effects of a high fat/high cholesterol diet on memory and hippocampal markers in aged rats

High fat diets have detrimental effects on cognitive performance, and can increase oxidative stress and inflammation in the brain. The aging brain provides a vulnerable environment to which a high fat diet could cause more damage. We investigated the effects of a high fat/high cholesterol (HFHC) diet on cognitive performance, neuroinflammation markers, and phosphorylated Tau (p-Tau) pathological markers in the hippocampus of Young (4-month old) versus Aged (14-month old) male rats. Young and Aged male Fisher 344 rats were fed a HFHC diet or a normal control diet for 6 months. All animals underwent cognitive testing for 12days in a water radial arm maze to assess spatial and working reference memory. Hippocampal tissue was analyzed by immunohistochemistry for structural changes and inflammation, and Western blot analysis. Young and Aged rats fed the HFHC diet exhibited worse performance on a spatial working memory task. They also exhibited significant reduction of NeuN and calbindin-D28k immunoreactivity as well as an increased activation of microglial cells in the hippocampal formation. Western blot analysis of the hippocampus showed higher levels of p-Tau S202/T205 and T231 in Aged HFHC rats, suggesting abnormal phosphorylation of Tau protein following the HFHC diet exposure. This work demonstrates HFHC diet-induced cognitive impairment with aging and a link between high fat diet consumption and pathological markers of Alzheimer’s disease.

Vulnerability of calbindin, calretinin and parvalbumin in a transgenic/knock-in APPswe/PS1dE9 mouse model of Alzheimer disease together with disruption of hippocampal neurogenesis

The pathogenesis of Alzheimer disease (AD) is characterized by accumulation of β-amyloid protein in the brain (in both soluble and insoluble forms) and by the presence of intracellular neurofibrillary tangles (NFTs), leading to neurotoxicity. The exact mechanisms whereby Aβ triggers brain alterations are unclear. However, accumulating evidence suggests that a deregulation of Ca2+ signaling may play a major role in disease progression. Calcium-buffering proteins, including calbindin-D28K (CB), calretinin (CR) and parvalbumin (PV), may offer neuroprotection by maintaining calcium homeostasis. Although marked reductions in these proteins have been observed in the brains of mice and humans with AD, their contribution to AD pathology remains unclear. The aim of the present study was to analyze distribution patterns of CB+, CR+ and PV+ interneurons in different areas of the hippocampus, a brain region that is severely affected in AD. A transgenic knock-in APPswe/PS1dE9 mouse model of familial AD was used. The data were obtained from the brains of 3- and 12-month-old animals. These ages roughly correspond to an early mature adult (prior to clinical manifestations) and a late middle-age (clinical symptoms readily detectable) phase in human AD patients. Immunostaining revealed increases in CB and PV immunoreactivity (IR) in the hippocampus of 3-month-old transgenic mice, compared to wild-type animals. Possibly, these proteins are upregulated in an attempt to control cellular homeostasis and synaptic plasticity. However, the pattern of CB-IR was reversed in 12-month-old animals, potentially indicating a loss of cellular capacity to respond to pathophysiological processes. In addition, at this age, a noticeable increase in PV-IR was observed, suggesting the presence of hippocampal network hyperactivity in older AD-like mice. Our results indicate that CaBP+ neuronal subpopulations play a role in adult neurogenesis and in AD pathology, particularly at early disease stages, suggesting that these neurons may serve as potential predictors of future AD in non-demented individuals.

Calbindin-D28k immunoreactivity in the mice thoracic spinal cord after space flight

AbstractThe aim of the work was to analyse changes in the location and morphological characteristics of calbindin (CB)-immunoreactive (IR) neurons of the thoracic spinal cord of C57BL/6N male mice after completion of a 30-day space flight on board the BION-M1 biosatellite (Russia, 2013). Space flight induced multidirectional changes of the number and morphological parameters of CB-positive neurons. The number of IR neurons increased in laminae I (from 10 to 17 neurons per section), II (from 42 to 67 cells per section) and IX (from two neurons per segment to two neurons per section), but CB disappeared in neurons of lamina VIII. Weightlessness did not affect the number of CB-IR neurons in laminae III–V and VII, including preganglionic sympathetic neurons. The cross-sectional area of CB-IR neurons decreased in lamina II and VII (group of partition cells) and increased in laminae III–V and IX. After a space flight, few very large neurons with long dendrites appeared in lamina IV. The results obtained give evidence about substantial changes in the calcium buffer system and imbalance of different groups of CB-IR neurons due to reduction of afferent information under microgravity.

Paraneoplastic CDR2 and CDR2L antibodies affect Purkinje cell calcium homeostasis.

Paraneoplastic cerebellar degeneration (PCD) is characterized by loss of Purkinje cells (PCs) associated with progressive pancerebellar dysfunction in the presence of onconeural Yo antibodies. These antibodies recognize the cerebellar degeneration-related antigens CDR2 and CDR2L. Response to PCD therapy is disappointing due to limited understanding of the neuropathological mechanisms. Here, we report the pathological role of CDR antibodies on the calcium homeostasis in PCs. We developed an antibody-mediated PCD model based on co-incubation of cerebellar organotypic slice culture with human patient serum or rabbit CDR2 and CDR2L antibodies. The CDR antibody-induced pathology was investigated by high-resolution multiphoton imaging and biochemical analysis. Both human and rabbit CDR antibodies were rapidly internalized by PCs and led to reduced immunoreactivity of calbindin D28K (CB) and L7/Pcp-2 as well as reduced dendritic arborizations in the remaining PCs. Washout of the CDR antibodies partially recovered CB immunoreactivity, suggesting a transient structural change in CB calcium-binding site. We discovered that CDR2 and CB co-immunoprecipitate. Furthermore, the expression levels of voltage-gated calcium channel Cav2.1, protein kinase C gamma and calcium-dependent protease, calpain-2, were increased after CDR antibody internalization. Inhibition of these signaling pathways prevented or attenuated CDR antibody-induced CB and L7/Pcp-2 immunoreactivity loss, morphological changes and increased protein expression. These results signify that CDR antibody internalization causes dysregulation of cell calcium homeostasis. Hence, drugs that modulate these events may represent novel neuroprotective therapies that limit the damaging effects of CDR antibodies and prevent PC neurodegeneration.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-014-1351-6) contains supplementary material, which is available to authorized users.

A new humanized ataxin-3 knock-in mouse model combines the genetic features, pathogenesis of neurons and glia and late disease onset of SCA3/MJD

Spinocerebellar ataxia type 3 (SCA3/MJD) is a neurodegenerative disease triggered by the expansion of CAG repeats in the ATXN3 gene. Here, we report the generation of the first humanized ataxin-3 knock-in mouse model (Ki91), which provides insights into the neuronal and glial pathology of SCA3/MJD. First, mutant ataxin-3 accumulated in cell nuclei across the Ki91 brain, showing diffused immunostaining and forming intranuclear inclusions. The humanized allele revealed expansion and contraction of CAG repeats in intergenerational transmissions. CAG mutation also exhibited age-dependent tissue-specific expansion, which was most prominent in the cerebellum, pons and testes of Ki91 animals. Moreover, Ki91 mice displayed neuroinflammatory processes, showing astrogliosis in the cerebellar white matter and the substantia nigra that paralleled the transcriptional deregulation of Serpina3n, a molecular sign of neurodegeneration and brain damage. Simultaneously, the cerebellar Purkinje cells in Ki91 mice showed neurodegeneration, a pronounced decrease in Calbindin D-28k immunoreactivity and a mild decrease in cell number, thereby modeling the degeneration of the cerebellum observed in SCA3. Moreover, these molecular and cellular neuropathologies were accompanied by late behavioral deficits in motor coordination observed in rotarod and static rod tests in heterozygous Ki91 animals. In summary, we created an ataxin-3 knock-in mouse model that combines the molecular and behavioral disease phenotypes with the genetic features of SCA3. This model will be very useful for studying the pathogenesis and responses to therapy of SCA3/MJD and other polyQ disorders.

Immunolocalization of Ephexin-1 in the Developing Canine Cerebellum

Ephexin-1 functions as guanine nucleotide exchange factors for the Rho-type GTPases which have important roles in neuronal development including axon guidance, migration, morphogenesis, and plasticity of neurons. As little is known about ephexin-1 in the cerebellum, we investigated the immunolocalization of ephexin-1 in the developing canine cerebellum. While the cellular maturation was followed by the temporal pattern, the calbindin D-28k and ephexin-1 immunoreactivities gradually increased in developing canine cerebellum. When compared to the calbindin D-28k immunoreactivities, belated ephexin-1 immunolocalization was observed in the Purkinje cells which aligned a single layer during cerebellar development. These results suggest that ephexin-1 might play an important role in the development of the Purkinje cells during the first two postnatal weeks based on its immunolocalization in the present study.

Development of the sexually dimorphic nucleus of the preoptic area and the influence of estrogen-like compounds.

One of the well-defined sexually dimorphic structures in the brain is the sexually dimorphic nucleus, a cluster of cells located in the preoptic area of the hypothalamus. The rodent sexually dimorphic nucleus of the preoptic area can be delineated histologically using conventional Nissl staining or immunohistochemically using calbindin D28K immunoreactivity. There is increasing use of the bindin D28K-delineated neural cluster to define the sexually dimorphic nucleus of the preoptic area in rodents. Several mechanisms are proposed to underlie the processes that contribute to the sexual dimorphism (size difference) of the sexually dimorphic nucleus of the preoptic area. Recent evidence indicates that stem cell activity, including proliferation and migration presumably from the 3rd ventricle stem cell niche, may play a critical role in the postnatal development of the sexually dimorphic nucleus of the preoptic area and its distinguishing sexually dimorphic feature: a signifi-cantly larger volume in males. Sex hormones and estrogen-like compounds can affect the size of the sexually dimorphic nucleus of the preoptic area. Despite considerable research, it remains un-clear whether estrogen-like compounds and/or sex hormones increase size of the sexually dimor-phic nucleus of the preoptic area via an increase in stem cell activity originating from the 3rd ventricle stem cell niche.

Selegiline (deprenyl) decreases calbindin-D28k expression in cortical neurons of rats socially deprived during the post-weaning period

Preclinical studies indicate that selegiline (deprenyl), frequently used in some neurodegenerative diseases, exert protective effects on central nervous system neurons of individuals exposed to social isolation (SI). Furthermore, it has been suggested that SI produces neuronal dysfunction due in part to an excessive intracellular Ca2+ overload. Since the main intracellular Ca2+ buffering mechanism involves changes in the calcium-binding protein calbindin-D28k (CB), and that CB neuronal expression can increase in response to Ca2+ transients, we hypothesized that chronic selegiline administration in early socially isolated animals could minimize cell CB expression as an indirect indicator of protective mechanism against Ca2+ overload. In the present study male rats were weaned at postnatal day 21 (P21) and randomly assigned to social deprivation (SI) or control (SC) environments for 30 days (P21–51). SI animals were further subdivided in two experimental groups: socially deprived-saline (SI-SAL) and socially isolated-selegiline (SI-SEL) for additional 30 days (P52–82). Medial frontal CB immunoreactivity (CB-ir) neurons were quantitatively and qualitatively analyzed. The results obtained indicate that neocortical cells of adult rats submitted to early SI show a significant increase in the number of CB-ir neurons per cortical field, while selegiline treatment significantly reduces this parameter.

In vivo AAV-mediated expression of calbindin-D28K in rat basal forebrain cholinergic neurons

The cholinergic neurons of the basal forebrain (BFCNs) in human and non-human primates are rich in the calcium binding protein calbindin-D28k (CB). We have shown a selective loss of CB from BFCNs in the course of normal aging, which appears to predispose these neurons to tangle formation and degeneration in Alzheimer's disease. Our previous preliminary investigation demonstrated that rodent BFCNs are devoid of CB. Here we confirm that rat choline acetyltransferase-rich BFCNs are devoid of CB immunoreactivity. We then describe a method for adeno-associated viral vector (AAV) induced expression of CB in rat BFCNs in vivo. We constructed AAV vectors bearing the CB gene under the control of the CMV promoter, or neuron-specific enolase (NSE) promoter, to bias expression in neurons. Both vectors resulted in CB expression in mouse neuronal cultures, and in rat brain following injections. AAV–NSE–CB resulted in more robust expression in neurons. Injections of 10μl of AAV–NSE–CB in the BFCNs component located within the internal segment of globus pallidus and internal capsule resulted in expression of CB in 84% of BFCNs. Expression was optimum at 14 days. Injections of AAV–NSE–LacZ resulted in robust β-galactosidase expression, but no CB immunoreactivity. Our results show that use of NSE promoter leads to high expression of genes in neurons and that the BFCNs can be targeted for expression of genes that are differentially expressed in the rodent and primate brains. These findings have important implications for gene replacement therapy in human BFCNs.

Indicators for acute hypoxia—An immunohistochemical investigation in cerebellar Purkinje-cells

The Purkinje-cells (PCs) of the cerebellum are highly vulnerable to hypoxic/ischemic insult. Calbindin-D28k is a calcium-binding protein that is strongly expressed in PCs. Following hypoxia, a decrease in its concentration has been found in animal models before any morphological change of the PCs took place. Hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) are increasingly expressed in tissues that undergo durations of hypoxia, and also in brain tissues.We examine whether a change in expression of any of these factors, or a combination of alterations, is an indicator of acute hypoxia. We investigated the intensity of neuronal immunoreactivity of calbindin-D28k, HIF-1α and VEGF retrospectively in 141 samples of human cerebellar tissue obtained from autopsies performed at the Institute of Legal Medicine of Hanover Medical School in 2007 and 2008. Three groups were compared. The first group comprises individuals (n=48) who died due to acute hypoxia, such as drowning or asphyxia. The second is a control group comprising individuals who died due to heart failure (n=56), and the third group comprised individuals who died almost instantly of polytraumata (n=37).Our study finds a statistically significant decrease in the expression of calbindin-D28k (p<0.05) in PCs in the acute hypoxia group, relative to the control groups. No changes in the expression of HIF-1α or in the expression of VEGF were observed in any of the groups. Consequently HIF-1α and VEGF were not suitable indicators, whereas detection of a decreasing concentration of calbindin-D28k supports a diagnosis of acute hypoxia.

Low oral doses of bisphenol A increase volume of the sexually dimorphic nucleus of the preoptic area in male, but not female, rats at postnatal day 21

Perinatal treatment with relatively high doses of bisphenol A (BPA) appears to have little effect on volume of the rodent sexually dimorphic nucleus of the preoptic area (SDN-POA). However, doses more relevant to human exposures have not been examined. Here, effects of pre- and post-natal treatment with low BPA doses on SDN-POA volume of postnatal day (PND) 21 Sprague–Dawley rats were evaluated. Pregnant rats were orally gavaged with vehicle, 2.5 or 25.0μg/kg BPA, or 5.0 or 10.0μg/kg ethinyl estradiol (EE2) on gestational days 6–21. Beginning on the day after birth, offspring were orally treated with the same dose their dam had received. On PND 21, offspring (n=10–15/sex/group; 1/sex/litter) were perfused and volume evaluation was conducted blind to treatment. SDN-POA outline was delineated using calbindin D28K immunoreactivity. Pairwise comparisons of the significant treatment by sex interaction indicated that neither BPA dose affected female volume. However, females treated with 5.0 or 10.0μg/kg EE2 exhibited volumes that were larger than same-sex controls, respectively (p<0.001). Males treated with either BPA dose or 10.0μg/kg/day EE2 had larger volumes than same-sex controls (p<0.006). These data indicate that BPA can have sex-specific effects on SDN-POA volume and that these effects manifest as larger volumes in males. Sensitivity of the methodology as well as the treatment paradigm was confirmed by the expected EE2-induced increase in female volume. These treatment effects might lead to organizational changes within sexually dimorphic neuroendocrine pathways which, if persistent, could theoretically alter adult reproductive physiology and socio-sexual behavior in rats.