Increase In Immunolabeling Research Articles

Characteristics of proliferation and apoptosis of hepatocytes after administration of ascorbic acid in a model of radiation hepatitis

BACKGROUND: Radiation hepatitis with the development of radiation-induced acute liver failure is considered one of the most serious complications of radiotherapy for malignant neoplasms of the liver, abdominal organs, or whole body irradiation. However, the exact mechanisms of radiation-induced liver cell death have not been fully elucidated, and therefore the study of changes in the proliferative-apoptotic ratio in liver structures remains relevant, and pre-irradiation administration of ascorbic acid can potentially protect them from the effects of electron irradiation.
 AIM: assessment of proliferation and apoptosis of hepatocytes after administration of ascorbic acid in a model of radiation hepatitis.
 METHODS: Wistar rats (Rattus Wistar; n=40) were divided into four experimental groups: I – control (n=10); II (n=10) – fractional irradiation with electrons in a total irradiation dose of 30 Gy; III (n=10) – administration of ascorbic acid before electron irradiation; IV (n=10) – administration of ascorbic acid. Animals of all groups (I – IV) were removed from the experiment a week after the last fraction. Morphological and immunohistochemical (with antibodies to Ki-67 and caspase-3) studies were carried out.
 RESULTS: A week after electron irradiation, a sharp decrease in the proportion of Ki-67-positive hepatocytes in combination with an increase in immunolabeling with antibodies to caspase-3 was observed in group II. During the administration of ascorbic acid in group III, less pronounced depth and range of liver damage was noted, confirmed by morphological and immunohistochemical methods (less pronounced decrease in the level of Ki-67 expression and an increase in the proportion of caspase-positive hepatocytes compared to the control) methods.
 CONCLUSION: An immunohistochemical study of proliferation and apoptosis of hepatocytes revealed that a week after fractional electron irradiation in SOD 30 Gy, there is a decrease in mitotic activity and an increase in cell death, and pre-irradiation administration of ascorbic acid helped level out the detected changes, which indicates its protective effect.

Acute restraint stress alters food-foraging behavior in rats: Taking the easier Way while suffered

Stress can influence decision-making in humans from many cognitive perspectives, while the underlying neurobiological mechanism remains incompletely understood. Food-foraging is a rodent behavior involving strategic possessing of nutritional supply in social context; experimental model of this behavior could help explore the effect of stress on decision-making and the brain mechanism thereof. In the present study, the influence of stress on food-foraging behavior was assessed in rats using an open field choosing paradigm wherein food collection (standard food or sweet food) were associated with social competition (with or without a rat in the cage). Acute restraint stress (ARS) was induced by placing the rat in a plastic restrainer for 2 h before food-foraging behavioral tests, with the effect of stress also determined biochemically and immunohistochemically. Restraint stressed rats showed anxiety-like behavior and elevation of serum corticosterone (CORT) and epinephrine (EPI) relative to controls. Both restraint and control animals preferred sugared food. However, the former group tended to forage food from a cage not occupied by a conspecific rat, whereas the control rats preferred to obtain food from the cage with a social competitor. Thus, the total amount of food foraged and eaten are reduced in the restrained rats than in controls. While the restraint animals had normal social interaction with other rats, they displayed enhanced social agonistic behavior. In brain examination, ARS attenuated the increase in immunolabeling and protein levels of c-fos, p-CREB, p-ERK1/2 in the anterior cingulate cortex (ACC) observed in control animals in association with food-foraging. These results indicate that restraint stressed rats tend to forage food by taking the advantage of a less competitive opportunity. Mechanistically, this decision-making alternative appears to be mediated through a neuronal deactivation in the ACC. The current findings provide novel insights into neuronal processing of decision-making behavior under the influence of stress.

3D Microfibrous Scaffolds Selectively Promotes Proliferation and Glial Differentiation of Adult Neural Stem Cells: A Platform to Tune Cellular Behavior in Neural Tissue Engineering.

Biomaterials are essential for the development of innovative biomedical and therapeutic applications. Biomaterials-based scaffolds can influence directed cell differentiation to improve cell-based strategies. Using a novel microfluidics approach, poly (ε-caprolactone) (PCL), is used to fabricate microfibers with varying diameters (3-40µm) and topographies (straight and wavy). Multipotent adult rat hippocampal stem/progenitor cells (AHPCs) are cultured on 3D aligned PCL microfibrous scaffolds to investigate their ability to differentiate into neurons, astrocytes, and oligodendrocytes. The results indicate that the PCL microfibers significantly enhance proliferation of the AHPCs compared to control, 2D planar substrates. While the AHPCs maintained their multipotent differentiation capacity when cultured on the PCL scaffolds, there is a significant and dramatic increase in immunolabeling for astrocyte and oligodendrocyte differentiation when compared with growth on planar surfaces. Our results show a 3.5-fold increase in proliferation and 23.4-fold increase in astrocyte differentiation for cells on microfibers. Transplantation of neural stem/progenitor cells within a PCL microfiber scaffold may provide important biological and topographic cues that facilitate the survival, selective differentiation, and integration of transplanted cells to improve therapeutic strategies.

Endogenous cholinergic neurotransmission contributes to behavioral sensitization to morphine.

Neuroplasticity in the mesolimbic dopaminergic system is critical for behavioral adaptations associated with opioid reward and addiction. These processes may be influenced by cholinergic transmission arising from the laterodorsal tegmental nucleus (LDTg), a main source of acetylcholine to mesolimbic dopaminergic neurons. To examine this possibility we asked if chronic systemic morphine administration affects expression of genes in ventral and ventrolateral periaqueductal gray at the level of the LDTg using rtPCR. Specifically, we examined gene expression changes in the area of interest using Neurotransmitters and Receptors PCR array between chronic morphine and saline control groups. Analysis suggested that chronic morphine administration led to changes in expression of genes associated, in part, with cholinergic neurotransmission. Furthermore, using a quantitative immunofluorescent technique, we found that chronic morphine treatment produced a significant increase in immunolabeling of the cholinergic marker (vesicular acetylcholine transporter) in neurons of the LDTg. Finally, systemic administration of the nonselective and noncompetitive neuronal nicotinic antagonist mecamylamine (0.5 or 2 mg/kg) dose-dependently blocked the expression, and to a lesser extent the development, of locomotor sensitization. The same treatment had no effect on acute morphine antinociception, antinociceptive tolerance or dependence to chronic morphine. Taken together, the results suggest that endogenous nicotinic cholinergic neurotransmission selectively contributes to behavioral sensitization to morphine and this process may, in part, involve cholinergic neurons within the LDTg.

C-Fos positive nucleus reveals that contextual specificity of latent inhibition is dependent of insular cortex

The present study analyzed the functional activity of granular and agranular insular cortices in contextual specificity of latent inhibition using a conditioned taste aversion paradigm. c-Fos immunolabeling was examined in insular cortex in preexposed and no preexposed groups under similar and different context conditions. Result showed that the exposition to a novel taste increased c-fos activity in insular cortex. However, a context shift caused an increase in immunolabeling in animals preexposed to saccharine. These results suggest insular cortex is part of a complex system to evaluate taste-response, and it may read the meaning of taste stimuli depending on the context.

S.04.01 Differential role of the GABA transporters in the regulation of extracellular concentrations of GABA in the rat hippocampus

In the adult cerebral cortex, GABA transporters (GATs) are expressed by both neurons and astrocytes. GAT-1 immunoreactivity is found in axon terminals of GABAergic neurons and astrocytes, while GAT-3 immunolabeling occurs only in the latter. The present study was designed to determine whether the expression of GAT-1 and GAT-3 in the adult rat cerebrum changes after needle lesion and colchicine infusion. Following a needle puncture or a saline injection, immunolabeling for GAT-1 and GAT-3 was slightly increased in an area around the needle track. Not only was the neuropil labeling for both GATs increased, but also a few neuronal somata were found to be immunoreactive for GAT-1. Colchicine injections induced a striking increase in immunolabeling for both GATs in the neuropil in an area adjacent to the needle path and surrounding it. A homologous region of the contralateral hemisphere also showed a moderate increase of immunoreactivity in the neuropil for both GATs. Furthermore, this contralateral site showed many neuronal somata immunolabeled for GAT-1. These changes were mainly detected during the first 5 days following intracortical lesions. These results indicate that (1) the upregulation of GAT-1 and GAT-3 in cortical interneurons and astrocytes is caused by both mechanical and chemical factors associated with the injections; (2) increased GAT-1 and GAT-3 expression contralateral to the site of colchicine injection is mediated by transcellular signaling across the corpus callosum; and (3) the lesion-induced GAT expression may play a protective role by helping to balance excitatory and inhibitory neuronal activities.

Increased expression of the transcription factor Prrxl1/Drg11 in dorsal root ganglia following peripheral inflammation in adult mice

The paired-type homeodomain transcription factor Prrxl1/Drg11 is a key regulator of the differentiation and survival of nociceptive neurons in pre and perinatal stages. Drg11-/- mice were shown to exhibit abnormalities in DRG spinal projections, defects in superficial dorsal horn structure and neurochemistry and reduced nociceptive behaviour in several pain tests. Here, we examined if the expression of Drg11 gene was affected by the induction of inflammatory pain. Peripheral inflammatory pain was induced by hindpaw plantar injection of zymosan in 21 days old male mice. After 4 hours the animals were sacrificed and L4-L5 DRGs ipsi and contralateral to the injection were removed for analysis of gene expression using Real Time PCR. Relative quantification of Drg11 gene expression was compared between ipsi- and contralateral DRGs. In addition, the amount of Drg11 expression was quantified by western blotting analysis and qualitative evaluation of Prrxl1-expressing neurons was also performed using immunohistochemistry analysis. Following zymosan injection Prrxl1 expression was 2.29 +/- 0.50 fold higher in the ipsilateral DRGs relative to the contralateral DRGs. Similar results were obtained by blotting analysis. Moreover, we observed an increase in immunolabeling of Drg11-positive DRG neurons of the side ipsilateral to the zymosan injection. The importance of Drg11 in the early development of nociceptive DRG and spinal cord neurons is well established. The present study shows for the first time that peripheral inflammation enhances the post-developmental level of Drg11 expression, and raises the possibility that this transcription factor may have a role in pain processing in adult mice. The paired-type homeodomain transcription factor Prrxl1/Drg11 is a key regulator of the differentiation and survival of nociceptive neurons in pre and perinatal stages. Drg11-/- mice were shown to exhibit abnormalities in DRG spinal projections, defects in superficial dorsal horn structure and neurochemistry and reduced nociceptive behaviour in several pain tests. Here, we examined if the expression of Drg11 gene was affected by the induction of inflammatory pain. Peripheral inflammatory pain was induced by hindpaw plantar injection of zymosan in 21 days old male mice. After 4 hours the animals were sacrificed and L4-L5 DRGs ipsi and contralateral to the injection were removed for analysis of gene expression using Real Time PCR. Relative quantification of Drg11 gene expression was compared between ipsi- and contralateral DRGs. In addition, the amount of Drg11 expression was quantified by western blotting analysis and qualitative evaluation of Prrxl1-expressing neurons was also performed using immunohistochemistry analysis. Following zymosan injection Prrxl1 expression was 2.29 +/- 0.50 fold higher in the ipsilateral DRGs relative to the contralateral DRGs. Similar results were obtained by blotting analysis. Moreover, we observed an increase in immunolabeling of Drg11-positive DRG neurons of the side ipsilateral to the zymosan injection. The importance of Drg11 in the early development of nociceptive DRG and spinal cord neurons is well established. The present study shows for the first time that peripheral inflammation enhances the post-developmental level of Drg11 expression, and raises the possibility that this transcription factor may have a role in pain processing in adult mice.

Ablation of Cx47 in transgenic mice leads to the loss of MUPP1, ZONAB and multiple connexins at oligodendrocyte–astrocyte gap junctions

Oligodendrocytes in CNS are linked to astrocytes by heterotypic gap junctions composed of Cx32 and Cx47 in oligodendrocytes and Cx30 and Cx43 in astrocytes. These gap junctions also harbour regulatory proteins, including ZO-1 and ZONAB. Here, we investigated the localization of multi-PDZ domain protein 1 (MUPP1) at these gap junctions and examined accessory proteins and connexins associated with oligodendrocytes in Cx47-knockout mice. In every CNS region tested, punctate immunolabelling for MUPP1 was found on all oligodendrocyte somata in wild-type mice. These MUPP1-positive puncta were colocalized with punctate labelling for oligodendrocytic Cx32 or Cx47, and with astrocytic Cx30 or Cx43 at oligodendrocyte-astrocyte (O/A) gap junctions, but were not found at astrocyte-astrocyte gap junctions. In Cx47-knockout mice, immunolabelling of MUPP1 and ZONAB was absent on oligodendrocytes, whereas some ZO-1-positive puncta remained. In Cx32-knockout mice, MUPP1 and ZONAB persisted at O/A gap junctions. The absence of Cx47 in Cx47-knockout mice was accompanied by a total loss of punctate labelling for Cx30, Cx32 and Cx43 on oligodendrocyte somata, and by a dramatic increase in immunolabelling for Cx32 along myelinated fibers. These results demonstrate MUPP1 at O/A gap junctions and Cx47-dependent targeting of connexins to the plasma membranes of oligodendrocyte somata. Further, it appears that deficits in myelination reported in Cx47-knockout mice may arise not only from a loss of Cx47 but also from the accompanied loss of gap junctions and their regulatory proteins at oligodendrocyte somata, and that loss of Cx47 may be partly compensated for by elevated levels of Cx32 along myelinated fibers.

MMP-13 (collagenase 3) immunolocalisation during initial orthodontic tooth movement in rats

Matrix metalloproteinases (MMPs) are enzymes that play a central role in periodontal ligament (PDL) space remodelling during orthodontic tooth movement. It has previously been shown that messenger RNA levels of MMP-13 increase significantly following the application of orthodontic forces. The aim of the present study was to examine immunolocalisation of MMP-13 and to evaluate if this collagenase is time-dependently and differentially detected within the PDL following the application of orthodontic forces to create areas of compression and tension. This was achieved by placing elastic bands between the maxillary first and second molars of 16 male Sprague-Dawley rats (each weighing 120-200g) for 12 and 24h. The molar-bearing segments were dissected and processed for histological and immunohistochemical examination. Binding of a monoclonal antibody was used to evaluate MMP-13 localization using an indirect streptavidin/biotin immunperoxidase technique. MMP-13 was found to be inducible at the protein level by the application of forces. The PDL and osteoblast-lineage cells showed a time-dependent increase in immunolabelling of MMP-13. Immunolabelling of MMP-13 was detected initially on the compression side, and then on both the compression and the tension sides. Since this increase in MMP-13 immunolabelling occurred very early following the application of an orthodontic force in both PDL and alveolar bone, this would indicate that MMP-13 might play an important role during tooth movement.

Early activation, motility, and homing of neonatal microglia to injured neurons does not require protein synthesis

Neuronal injury in CNS tissues induces a rapid activation and mobilization of resident microglia (MG). It is widely assumed that changes in gene expression drive the morphological transformation of MG and regulate their mobilization during activation. Here, we used acutely excised neonatal rat brain slices to test whether the morphological transformation and homing of MG to injured neurons requires gene expression and de novo protein synthesis. Traumatic injury during excision of live brain tissue slices induces a rapid and transient translocation of a transcription factor, NF-kappaB, to nuclei in MG. This is followed within 4-8 h by an increase in immunolabeling for cell adhesion molecules and lysosomal proteins, accompanied by changes in cell morphology. Application of anisomycin, a protein synthesis inhibitor, prevents the increase in immunolabeling for markers of MG activation but not the morphological transformation. Confocal time-lapse imaging in live tissue slices indicates that MG cell motility (branch extension and retraction) and locomotion are unaffected by anisomycin at early postinjury time-points (<4 h), while at later time-points (4-8 h postinjury) MG locomotion but not motility is inhibited. Thus, activated MG rapidly localize to injured pyramidal neuron cell bodies by 4-h postinjury, even in the presence of anisomycin. Moreover, this early MG activation and homing to injured neurons is unaffected in tissue slices from beta2 integrin deficient mice. These results indicate that gene activation and new protein synthesis coincide with, but are not necessary for, the rapid morphological transformation and early migration-dependent homing of activated MG to injured neurons in CNS tissues.

Pregranulomatous phase of sarcoidosis: Immunohistochemical diagnosis

Histopathological confirmation of clinical suspicion of sarcoidosis is based on the finding of non-caseating granulomas in biopsy material, usually in prescalene lymph nodes or in transbronchial lung biopsies. Lymph node reactive sinus histiocytosis (RSH) seen in relation to various inflammatory and non-inflammatory diseases can mimic the pregranulomatous phase of sarcoidosis (PSH). Differentiation of sinus histiocytosis based on histopathological features alone is limited. The purpose of this study is immunohistochemical determination of lymph node cellular response in granulomatous sarcoidosis, the PSH and RSH using a immunohistochemistry employing a panel of antibodies. Patient groups under study each contained 25 patients and included: those with clinical picture of sarcoidosis and non-caseating granulomatous lymphadenitis; those with confirmed sarcoidosis and with sinus histiocytosis without granuloma formation in lymph nodes; and finally, those without sarcoidosis and with "reactive" sinus histiocytosis in lymph nodes. Lymph node biopsy tissue was fixed in buffered formaldehyde, routinely processed to paraffin wax blocks, cut into 4-microm-thick sections, stained with hematoxylin and eosin and immunohistochemically labelled using a triple-layer APAAP protocol with purified polyclonal antibodies directed against SP 70 and SP90 from Mycobacterium tuberculosis and monoclonal antibodies against CD22, CD4, CD8, CD56, and CD68. Intensity of immunolabelling was assessed semiquantitatively by two independent observers. An increased CD4:CD8 ratio, moderate increase of immunolabelling for CD68 and slight decrease in immunolabelling for CD20, CD56, and SP90 was indicative of PSH when compared with RSH. The most notable difference between the studied groups was a difference in immunoreactivity to SP70 and CD4 antibodies. Lymph nodes with pregranulomatous sinus histiocytosis labelled with both antibodies. This profile of immunolabelling can be used in the differentiation of this condition from reactive sinusoidal lesions.

The transcription factor c-jun is activated in retinal ganglion cells in experimental rat glaucoma

This study investigates the role of the MAP kinase pathway including c-jun, ATF-2 and JNK in glaucomatous eyes of rats and in optic nerve transection. Glaucoma was induced in one eye of 51 adult Wistar rats by laser treatment to the trabecular meshwork. Eighteen further rats underwent unilateral optic nerve transection. We studied the transcription factor c-jun, its activated form, phospho-c-jun, the transcription factor p-ATF-2, and the enzyme JNK by immunohistochemistry. The activation of p-c-jun was also investigated using western blot analysis. Treated and control eyes were compared in a masked way at multiple time points after injury. We found a statistically significant increase in immunolabelling for c-jun and phospho-c-jun in retinal ganglion cells (RGCs) from 1 day to 4 weeks after intraocular pressure (IOP) elevation. At 1 and 2 days after the laser treatment, a mean of 2.9±3.3 RGCs mm −1 were positive for c-jun ( n=12, p=0.005, t-test), increasing to a mean of 13.4±7.5 cells mm −1 at 1 week ( n=18, p=0.00005), and decreasing to 2.3±2.0 cells mm −1 at 2 weeks ( n=5, p=0.04) and 0.1±0.1 cells mm −1 at 2 months. Few of the 47 control eyes had any labelling for c-jun or phospho-c-jun, while between 80 and 100% of elevated IOP eyes showed positivity during the first 2 weeks of experimental glaucoma. After optic nerve transection, c-jun and phospho-c-jun were also significantly activated at 1, 2 and 9 days ( p<0.03, t-test). Western blot analysis demonstrated significantly increased phospho-c-jun amounts in both transected and glaucomatous eyes compared to control fellow eyes 1 week following treatment. JNK was not significantly activated in glaucoma or optic nerve transection. P-ATF-2 was not significantly activated in glaucoma, but was significantly increased 2 days after optic nerve transection. We conclude that the process leading to RGC death in experimental glaucoma and after optic nerve transection involves the activation of c-jun at the RGC layer. C-jun is activated more gradually in glaucoma then after optic nerve transection.

Characteristic distribution of immunoreaction for estrogen receptor alpha in rat ameloblasts

Estrogen has a diverse function, including cell proliferation and differentiation via estrogen receptors (ER), which have been reported to be the case in various tissues in addition to female reproductive organs. A recent immunocytochemical study has reported the expression of ERalpha, a subtype of ER, in rat odontoblasts, suggesting an involvement of estrogen in the differentiation of tooth-forming cells. However, there is no information on the ERalpha immunoexpression in ameloblasts. The present study was therefore undertaken to examine the localization of ERalpha immunoreaction in rat ameloblasts during amelogenesis. A computer-assisted quantitative analysis under a confocal laser scanning microscope was employed to demonstrate the stage-specific localization pattern of ERalpha immunoreaction. Immunohistochemistry of the rat enamel organ revealed ERalpha expression as nuclear localization in ameloblasts, stratum intermedium, stellate reticulum, and papillary layer, in addition to mature and immature odontoblasts. The ratio of immunopositive nuclei to total nuclei (immunopositive ratio) in ameloblasts was high at the apical loop region and gradually declined at the presecretory stage to zero at the secretory stage with statistically significant difference. The ERalpha immunolabeling pattern exhibited a periodic change at the maturation stage proper with constant higher labeling in ruffle-ended ameloblasts than in smooth-ended ameloblasts. The positive ratio was then followed by a statistically significant increase in immunolabeling thereafter. This stage-specific immunolabeling pattern during amelogenesis suggests a possible role of ERalpha in ameloblast proliferation and differentiation.

Brain lactate synthesis in thiamine deficiency: A re‐evaluation using 1H‐13C nuclear magnetic resonance spectroscopy

Region-selective accumulation of brain lactate occurs in TD; however, the mechanisms responsible have not been elucidated fully. (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy were therefore used to investigate de novo lactate synthesis from [1-(13)C]glucose in vulnerable (medial thalamus) and nonvulnerable (frontal cortex) brain regions of rats made thiamine deficient by administration of the central thiamine antagonist pyrithiamine. De novo synthesis of lactate was increased in the medial thalamus to 148% and 226% of pair-fed control values at presymptomatic and symptomatic stages of thiamine deficiency, respectively, whereas no such changes were observed in the frontal cortex. Administration of a glucose load selectively worsened the changes in medial thalamus. Pyruvate recycling and peripherally derived lactate did not contribute significantly to the lactate increase within the thiamine-deficient brain. Increases in immunolabeling of the lactate dehydrogenase isoenzymes (LDH1 and LDH5) were observed in the medial thalamus of thiamine-deficient animals. Metabolic impairment due to thiamine deficiency thus results in increased glycolysis, increased LDH immunolabeling of neurons and astrocytes and increased de novo synthesis of lactate in brain regions vulnerable to thiamine deficiency. These results are consistent with the notion that focal lactate accumulation participates in the worsening of neurologic symptoms in thiamine-deficient patients.

Induction of bone marrow stromal cells to neurons: differentiation, transdifferentiation, or artifact?

Differentiation of stem cells toward a neuronal lineage normally involves a gradually progressive restriction in developmental potential and is regulated by a diverse set of specific and temporally precise genetic events. However, recent studies have indicated that both rodent and human bone marrow stromal cells (MSCs) can be rapidly (within minutes to hours) induced to differentiate into neurons in vitro by relatively simple chemical means (using beta-mercaptoethanol [BME] or dimethylsulfoxide [DMSO] and butylated hydroxyanisol [BHA]; Woodbury et al. [ 2000] J. Neurosci. Res. 61:364-370). The ability to transdifferentiate an easily accessible cell source into neurons could have substantial potential for promoting neural repair. We therefore explored the potential of simple chemical methods to transdifferentiate other cell types, including primary rat fibroblasts, primary human keratinocytes, HEK293 cells, rat PC-12 cells, and as positive control rat bone marrow stromal (BMS) cells. Surprisingly, all cells except for keratinocytes adopted at least partial "neuron-like" pyramidal cell morphology with fine-cellular extensions resembling neurites upon stimulation with BME or DMSO/BHA. However, time-lapse microscopy indicated that the chemical exposure of MSCs did not result in new neurite growth but rather cellular shrinkage, with retraction of the majority of existing cell extensions, leaving only few, fine neurite-like processes. To determine whether the chemically induced transdifferentiation resulted from simple cellular toxicity, MSCs were exposed to various stressors, including detergents, high-molarity sodium chloride, and extremes of pH. In all cases, cellular shrinkage and adoption of pseudoneuronal morphology were observed. Concomitantly with cellular shrinkage, apparent increases in immunolabeling for the neuronal markers NSE and NeuN were detected in the cell soma that could not be confirmed by RT-PCR. Furthermore, blockade of protein synthesis with cycloheximide did not prevent cells from adopting "neuron-like" morphology after chemical induction. Thus, morphological changes and increases in immunolabeling for certain cellular markers upon "chemical induction" of MSCs are likely the result of cellular toxicity, cell shrinkage, and changes in the cytoskeleton and do not represent regulated steps in a complicated cellular differentiation process.

Abnormal postnatal ontogeny of the locus coeruleus in the epileptic mutant mouse quaking

The tonic-clonic convulsions of the quaking mutant mice have been shown to be associated with the hyperplasia of the nucleus locus coeruleus, the origin of most brain noradrenergic neurons. In the present study, the postnatal ontogeny of the locus coeruleus has been studied by tyrosine hydroxylase immunolabeling in the mutant mice quaking and their controls at postnatal days 1, 30 and 90. In the control mice, the number of immunoreactive neuronal cell bodies increased significantly in the rostral half of the locus coeruleus between birth and postnatal day 30, while it decreased significantly in the caudal half between birth and adulthood. Thus, during postnatal maturation, the distribution of locus coeruleus neurons was shifted in the rostral direction. In the quaking mutant mice, while the increase of immunolabeling between birth and postnatal day 30 was observed in the rostral half of the locus coeruleus, no diminution could be found in the caudal half between birth and adulthood. As a result, the rostral shift of tyrosine hydroxylase immunoreactivity was not observed. Consequently, in adult mice, the caudal part of the mutants locus coeruleus appeared to contain significantly more neurons than the corresponding region in the controls. These results indicate that the hyperplasia of the locus coeruleus of the quaking mice that we had previously reported results from an alteration of the postnatal maturation of this nucleus. This developmental abnormality might be a primary determinant of the inherited epilepsy of the quaking mutant mice.

Polysialylated neural cell adhesion molecule is involved in the neuroplasticity induced by axonal injury in the avian ciliary ganglion.

We demonstrated previously in the quail ciliary ganglion, that the immunoreactivity for the neural cell adhesion molecule labeling the postsynaptic specializations of intraganglionic synapses decreases when synaptic remodeling is induced by crushing the postganglionic ciliary nerves. Here we show, in the same experimental conditions, that the immunolabeling for its polysialylated non-stabilizing isoform, which promotes cell plasticity, increases at these subcellular compartments. In control ganglia, poor immunolabeling for the polysialylated neural cell adhesion molecule was occasionally observed surrounding the soma of the ciliary neurons, in correspondence with the calyciform presynaptic ending and the perineuronal satellite cells sheath. At the electron microscope, several neuronal compartments, including some postsynaptic specializations, somatic spines and multivesicular bodies, were immunopositive. Three to six days after ciliary nerve crush, both the number of ciliary neurons labeled for the polysialylated neural cell adhesion molecule and the intensity of their immunolabeling increased markedly. Electron microscopy revealed that, in parallel to the injury-induced detachment of the preganglionic boutons, numerous postsynaptic specializations were found to be immunopositive. Twenty days later, when intraganglionic connections were re-established, polysialylated neural cell adhesion molecule immunoreactivity was comparable to that observed in control ganglia. The increase in immunolabeling also involved the other neuronal compartments mentioned above, the perineuronal satellite cells and the intercellular space between these and the ciliary neurons. From these results we suggest that the switch, at the postsynaptic specializations, between the neural cell adhesion molecule and its polysialylated form may be among the molecular changes occurring in axotomized neurons leading to injury-induced synaptic remodeling. Moreover, from the increase in polysialylated neural cell adhesion molecule immunolabeling, observed at the somatic spines and at the interface between neurons and perineuronal satellite cells, we suggest that this molecule may be involved not only in synaptic remodeling, but also in other more general aspects of injury-induced neuronal plasticity.

Distribution of Glutamate Receptor Subunit NMDAR1 in the Hippocampus of Normal Elderly and Patients with Alzheimer's Disease

Immunocytochemical techniques were employed to study the distribution and cytological features of NMDAR1-immunoreactive elements in the human hippocampal formation. Subjects with Alzheimer's disease (AD), presenting with a wide range of neuropathology and classified into six Braak stages (I–VI), and nondemented age-matched controls were examined. In control cases, the most intense NMDAR1 immunoreactivity was observed within the soma and dendrites of granule cells in the dentate gyrus and pyramidal neurons in Ammon's horn. Whereas small variations in the pattern of immunoreactivity were noted in control cases, AD subjects were characterized with intersubject variability which in most instances correlated with neuropathologic severity. For example, AD cases, particularly those with mild/modest pathology (Braak I–III), were indistinguishable from controls in the overall pattern of immunolabeling. In contrast, in those more severe AD cases (Braak IV–VI) the intensity of immunolabeling within the CA fields was greater than observed in controls and those with mild AD pathology. In addition, in pathologically severe cases numerous NMDAR1-positive pyramidal neurons were characterized by unique morphologic features including long and often tortuous apical dendrites. These latter findings were most prevalent in the CA1 region and subiculum. In contrast to the marked increase in immunolabeling in the CA fields, in the dentate gyrus we observed a reduction in NMDAR1 labeling particularly within the outer molecular layer (i.e., termination zone of the perforant pathway). This latter region was also the site of a number of NMDAR1-labeled plaques. Notably, the overall pattern of NMDAR1 immunoreactivity is distinct from that observed with antibodies against AMPA receptor subunits and suggests a differential role of various inotropic glutamate receptors in hippocampal plasticity in AD.

Fixative-Dependent Increase in Immunogold Labeling following Antigen Retrieval on Acrylic and Epoxy Sections

We examined the increase in immunogold labeling of variably fixed, resin embedded tissue sections following antigen retrieval by heating in citrate solution. Fibrin clots and porcine renal tissue were fixed in glutaraldehyde, paraformaldehyde or ethanol, and specimens were embedded in LR-White or epoxy resin. Immunogold labeling was performed on ultra-thin sections with anti-fibrinogen for the fibrin clots and anti-IgG for the porcine renal tissue. Immunogold labeling increased greatly after heating epoxy sections regardless of the fixative used. The ratio labelingretrieved/labelingnonretrieved (Lr/Ln) was 2.8 or higher, and the largest increases were obtained for anti-IgG. Heating induced a large increase of immunolabeling for LR-White sections only when the specimens had been fixed in paraformaldehyde (Lr/Ln = 2.2 for anti-IgG and 1.4 for antifibrinogen). LR-White sections showed decreased, insignificant or weakly increased immunolabeling of ethanol or glutaraldehyde fixed tissues following antigen retrieval. Disruption of aldehyde cross-links is not the only mechanism for antigen retrieval when epoxy sections are heated in citrate solution since large increases in immunolabeling were obtained on ethanol fixed tissue. The large heat-induced increases in immunolabeling on epoxy sections are probably caused by the disruption of chemical bonds between the epoxy resin and side groups of proteins.

Alteration of GABA transporter expression in the rat cerebral cortex following needle puncture and colchicine injection

In the adult cerebral cortex, GABA transporters (GATs) are expressed by both neurons and astrocytes. GAT-1 immunoreactivity is found in axon terminals of GABAergic neurons and astrocytes, while GAT-3 immunolabeling occurs only in the latter. The present study was designed to determine whether the expression of GAT-1 and GAT-3 in the adult rat cerebrum changes after needle lesion and colchicine infusion. Following a needle puncture or a saline injection, immunolabeling for GAT-1 and GAT-3 was slightly increased in an area around the needle track. Not only was the neuropil labeling for both GATs increased, but also a few neuronal somata were found to be immunoreactive for GAT-1. Colchicine injections induced a striking increase in immunolabeling for both GATs in the neuropil in an area adjacent to the needle path and surrounding it. A homologous region of the contralateral hemisphere also showed a moderate increase of immunoreactivity in the neuropil for both GATs. Furthermore, this contralateral site showed many neuronal somata immunolabeled for GAT-1. These changes were mainly detected during the first 5 days following intracortical lesions. These results indicate that (1) the upregulation of GAT-1 and GAT-3 in cortical interneurons and astrocytes is caused by both mechanical and chemical factors associated with the injections; (2) increased GAT-1 and GAT-3 expression contralateral to the site of colchicine injection is mediated by transcellular signaling across the corpus callosum; and (3) the lesion-induced GAT expression may play a protective role by helping to balance excitatory and inhibitory neuronal activities.