Light Microscopic Immunohistochemistry Research Articles

Increase of glutamate in satellite glial cells of the trigeminal ganglion in a rat model of craniofacial neuropathic pain.

Satellite glial cells (SGCs) that envelop the cell bodies of neurons in sensory ganglia have been shown to both release glutamate, and be activated by glutamate in the context of nociceptive signaling. However, little is known about the subpopulations of SGCs that are activated following nerve injury and whether glutamate mechanisms in the SGCs are involved in the pathologic pain. To address this issue, we used light and electron microscopic immunohistochemistry to examine the change in the glutamate levels in the SGCs and the structural relationship between neighboring neurons in the trigeminal ganglion (TG) in a rat model of craniofacial neuropathic pain, CCI-ION. Administration of ionomycin, ATP and Bz-ATP induced an increase of extracellular glutamate concentration in cultured trigeminal SGCs, indicating a release of glutamate from SGCs. The level of glutamate immunostaining in the SGCs that envelop neurons of all sizes in the TG was significantly higher in rats with CCI-ION than in control rats, suggesting that SGCs enveloping nociceptive as well as non-nociceptive mechanosensitive neurons are activated following nerve injury, and that the glutamate release from SGCs increases in pathologic pain state. Close appositions between substance-P (SP)-immunopositive (+) or calcitonin gene-related peptide (CGRP)+, likely nociceptive neurons, between Piezo1+, likely non-nociceptive, mechanosensitive neurons and SP+ or CGRP+ neurons, and between SGCs of neighboring neurons were frequently observed. These findings suggest that glutamate in the trigeminal SGCs that envelop all types of neurons may play a role in the mechanisms of neuropathic pain, possibly via paracrine signaling.

Bipolar cells containing protein kinase Cα mediate attentional facilitation of the avian retinal ganglion cells by the retinopetal signal.

Birds have a well-developed retinopetal system projecting from the midbrain to the contralateral retina. The signal sent to the retina through the retinopetal system facilitates visual responses of the retinal ganglion cells (RGCs), and the retinopetal signals function as attentional signals during visual search. Thus, the retinopetal signal somehow reaches and facilitates visual responses of the RGCs. However, the tertiary neuron of the retinopetal system, the isthmo-optic target cell (IOTC), is unlikely to contact most RGCs directly, because the IOTCs form axon terminals localized in the outermost sublayer (lamina 1) of the inner plexiform layer (IPL) where few RGC dendrites terminate. Therefore, some other intrinsic retinal neurons must be involved in the centrifugal attentional enhancement of visual responses of the RGCs. We investigated connections of the target cells of the IOTCs in chicken and quail, using light and electron microscopic immunohistochemistry. We show that axon terminals of the IOTC make synaptic contacts with protein kinase Cα (PKCα)-immunoreactive (ir) bipolar cells (PKCα-BCs) in lamina 1 of the IPL. Furthermore, with prolonged electrical stimulation of the isthmo-optic nucleus (ION) on one side, whose neurons send their axons to the contralateral retina and make synaptic contacts there with IOTCs, phosphorylation of cAMP response element-binding protein was observed in the PKCα-BCs in the contralateral retina, but not in the ipsilateral retina. This suggests that electrical stimulation of ION activated PKCα-BCs through synapses from IOTCs to PKCα-BCs, thus stimulating transcription in PKCα-BCs. Thus, centrifugal attentional signals may facilitate visual responses of RGCs via the PKCα-BCs.

The neuroprotective effect of ginsenoside Rb1 on the cerebral cortex changes induced by aluminium chloride in a mouse model of Alzheimer's disease: A histological, immunohistochemical, and biochemical study

Alzheimer's disease (AD) is one of the most common types of dementia among neurodegenerative disorders characterized by attention deficits and memory loss. Panax ginseng is a traditional Chinese herbal remedy that has been employed for millennia to manage dementia linked with aging and memory impairment. Ginsenoside Rb1 is one of Panax ginseng's most abundant components. The present work evaluated the neuroprotective effects of ginsenoside Rb1 on the cerebral cortex of AlCl3-induced AD in adult male albino mice. Forty male mice were alienated arbitrarily into; control group, ginsenoside Rb1 group (70 mg/kg/day), AlCl3 group (50 mg/kg/day), and ginsenoside Rb1-AlCl3 group that received ginsenoside Rb1 one hour before AlCl3. Oxidative stress parameters, Amyloid β (Aβ) and phosphorylated tau protein, and acetylcholine esterase (AChE) activity were measured. Cerebral cortex sections were evaluated histologically by light microscopic examination and immunohistochemistry. AlCl3-induced memory impairment, Aβ and phosphorylated tau protein accumulation, and AChE elevation. Moreover, histopathological alterations in the cerebral cortex were reported in the form of irregular shrunken neurons and the surrounding neuropil showed vacuolation. Some neurons appeared with darkly stained nuclei, others had faintly stained ones. The synaptophysin expression was significantly decreased, while the expression of cleaved caspase-3, glial fibrillary acidic protein (GFAP), and ionized calcium-binding adaptor molecule 1 (Iba-1) were significantly elevated. It's interesting to note that these changes were attenuated in mice pretreated with ginsenoside Rb1. Collected data indicated that ginsenoside Rb1 showed a potential neuroprotective effect against cerebral cortex changes caused by AlCl3 via suppression of Amyloid β and phosphorylated tau protein formation, oxidative stress correction, anti-apoptotic effect, and by minimizing gliosis.

Expression of Piezo2 in the Dental Pulp, Sensory Root, and Trigeminal Ganglion and Its Coexpression with Vesicular Glutamate Transporters

IntroductionInformation on the type of vesicular glutamate transporter (VGLUT) that is expressed in the Piezo2-positive (Piezo2+) neurons in the trigeminal ganglion (TG) and on the type of Piezo2+ axons and their distribution in the dental pulp is important for understanding dental pain elicited by mechanical stimuli and developing new therapeutic strategies. MethodsWe examined the expression of Piezo2 and its coexpression with VGLUT1 and VGLUT2 in rat TG, the sensory root, and human dental pulp using light and electron microscopic immunohistochemistry and quantitative analysis. ResultsVGLUT1 and VGLUT2 were expressed in the TG neurons. Piezo2 was expressed in axons of all types but primarily in small myelinated (Aδ) axons in the sensory root. In the dental pulp, Piezo2 was expressed densely in the numerous axons that form a plexus in the peripheral pulp. Piezo2+ axons in the peripheral pulp were mostly unmyelinated, and Piezo2 immunoreactivity was often concentrated near the axolemma, suggesting that it may represent functional receptors. ConclusionsThese findings suggest that VGLUT1 and VGLUT2 are involved in the glutamate signaling in Piezo2+ neurons, Piezo2 may be primarily activated by noxious mechanical stimuli, and Piezo2-mediated dental mechanotransduction may be primarily elicited in the peripheral pulp.

Expression of Piezo1 in the Trigeminal Neurons and in the Axons That Innervate the Dental Pulp.

Information on the neurons and axons that express the mechanosensitive channel Piezo1 and its expression in axons innervating the dental pulp may help understand the nature of the Piezo1-mediated mechanosensation and the underlying mechanism of dentin sensitivity elicited by mechanical stimuli. For this, we here investigated the neurochemical properties of the neurons in the rat trigeminal ganglion (TG) and their axons in its sensory root that express Piezo1 and the expression of Piezo1 in the rat and human dental pulp by light and electron microscopic immunohistochemistry and quantitative analysis. Piezo1 was expressed mainly in medium-sized and large TG neurons. Piezo1-immunopositive (+) neurons frequently coexpressed the marker for neurons with myelinated axons, NF200, but rarely the markers for neurons with unmyelinated axons, CGRP or IB4. In the sensory root of TG, Piezo1 was expressed primarily in small myelinated axons (Aδ, 60.2%) but also in large myelinated (Aβ, 24.3%) and unmyelinated (C, 15.5%) axons. In the human dental pulp, Piezo1 was expressed in numerous NF200+ axons, which formed a network in the peripheral pulp and often “ascended” toward the dentin. Most Piezo1+ myelinated axons in the radicular pulp became unmyelinated in the peripheral pulp, where Piezo1 immunoreaction product was associated with the axonal plasma membrane, suggesting a functional role of Piezo1 in the peripheral pulp. These findings suggest that Piezo1 is involved primarily in mediating the acute pain elicited by high-threshold mechanical stimuli, and that the Piezo1-mediated dental mechanotransduction occurs primarily in the axons in the peripheral pulp.

Retinoid receptors are expressed in mouse and human lungs.

Retinoid receptors are members of nuclear receptor superfamily consisting of two distinct families: RARs (retinoic acid receptors) and RXRs (retinoid X receptors). Each family contains three receptor subtypes α, β, and γ. Retinoids transduce their effects through binding to retinoid receptors and inhibit transcription factors such as activator protein-1 and nuclear factor-κB (NF-κB) both of which regulate the transcription of several inflammatory genes. Considering the role of retinoid receptors in lung physiology, we need a precise understanding of their expression in normal and inflamed lungs. We used light and electron microscopic immunohistochemistry and Western blot to determine the expression of retinoid receptors in a murine model of endotoxin-induced (E. coli; 055:B5, 80 μg intranasal) acute lung inflammation and normal human lungs. Western blot showed expression of all six retinoid receptor subtypes in normal and inflamed mouse lungs. Immunohistology localized differential expression of retinoid receptors in airway epithelium, alveolar/septal macrophages, vascular endothelium, and alveolar septum in mouse lungs. Intranasal LPS challenge in mice resulted in increased expression of RXRα in airway epithelium compared to control animals. All six retinoid receptor subtypes were expressed in normal human lungs. Immunoelectron microscopy further confirmed the localization of all the receptors in various lung cells including the nucleus of these cells. The basal and altered expression of retinoid receptors in normal and inflamed lungs, respectively, may suggest their roles in lung pathophysiology.

Retrospective immunohistochemical investigation of suspected non-visceral leiomyosarcoma in dogs.

Visceral leiomyosarcoma is well described in dogs, but information about non-visceral locations and prevalence is lacking. The diagnosis of leiomyosarcoma is challenging without a gold standard, and often includes the use of immunohistochemical (IHC) stains. We used defined histopathologic patterns, histochemical staining, and IHC staining for smooth muscle actin (SMA), desmin, and laminin to characterize suspected non-visceral leiomyosarcoma in dogs at a single academic institution. In a retrospective search, we identified 24 dogs with a definitive or suspected histologic diagnosis of leiomyosarcoma in a non-visceral location. Histopathology results and clinical details were obtained. Biopsy sections were reviewed by a single pathologist using standardized histologic criteria, including light microscopic appearance, immunohistochemistry (more than two-thirds of neoplastic cells labeled with SMA and desmin or laminin), and histochemical staining (minimal-to-mild matrix deposition by Masson trichrome). Of the 24 cases of possible non-visceral leiomyosarcomas, 4 were consistent with a definitive diagnosis of non-visceral leiomyosarcoma (3) or leiomyoma (1) based on the established criteria. Only the leiomyoma had more than two-thirds of neoplastic cells label with all 3 markers; all 3 leiomyosarcomas had more than two-thirds of neoplastic cells label with SMA and laminin. Our data highlight the uncommon nature of non-visceral leiomyosarcoma and the importance of IHC for their diagnosis. A definitive diagnosis could not be made based on SMA alone, and desmin was not useful in this cohort. Further studies are needed to clarify the histopathologic, IHC, and clinical features of canine non-visceral SMA-positive mesenchymal tumors.

Kv2.1 expression in giant reticular neurons of the postnatal mouse brain

Previous experiments charted the development of behavioral arousal in postnatal mice. From Postnatal Day 3 (P3) to Postnatal Day 6 (P6) mice (a) become significantly more active, “arousable”; and (b) in large reticular neurons, nucleus gigantocellularis (NGC), patch clamp recordings reveal a significantly increased ability to fire high frequency trains of action potentials as are associated with elevated cortical arousal. These action potential trains depend on delayed rectifiers such as Kv2.1. Here we report tracking the development of expression of a delayed rectifier, Kv2.1 in NGC neurons crucial for initiating CNS arousal. In tissue sections, light microscope immunohistochemistry revealed that expression of Kv2.1 in NGC neurons is greater at day P6 than at P3. Electron microscope immunohistochemistry revealed Kv2.1 labeling on the plasmalemmal surface of soma and dendrites, greater on P6 than P3. In brainstem reticular neuron cell culture, Kv2.1 immunocytochemistry increased monotonically from Days-In-Vitro 3–10, paralleling the ability of such neurons to fire action potential trains. The increase of Kv2.1 expression from P3 to P6, perhaps in conjunction with other delayed rectifier currents, could permit the ability to fire action potential trains in NGC neurons. Further work with genetically identified NGC neurons is indicated.

Blood-brain barrier leakage of blood proteins in idiopathic normal pressure hydrocephalus

AimIdiopathic normal pressure hydrocephalus (iNPH) is one subtype of dementia characterized by cerebrospinal fluid (CSF) disturbance, but with unknown cause. We recently reported that frontal cortex biopsies of iNPH patients disclosed degenerative alterations of the capillary basement membrane, including degenerated pericyte processes. Given that pericyte degeneration is associated with blood-brain barrier (BBB) dysfunction, the present study was undertaken to examine whether BBB leakage of blood proteins can be revealed by light microscopy (LM) immunohistochemistry in iNPH. MethodsThe study included cortical brain tissue specimens from 14 reference (REF) subjects undergoing neurosurgery for epilepsy, aneurysm or tumor, and 45 iNPH patients. Dysfunction of the BBB was measured semi-quantitatively as area percentage extravasated fibrin(ogen) in cerebral cortical layers I, II and III. The degree of fibrin(ogen) extravasation was also correlated with expression of glial fibrillary acidic protein (GFAP), aquaporin-4 (AQP4), dystrophin 71 (Dp71) and Cluster of Differentiation 68 (CD68). ResultsThe study disclosed extravasation of fibrin(ogen) in 4/14 REF subjects and in 45/45 iNPH patients, the percentage area of fibrin(ogen) was significantly higher in iNPH than REF cortical specimens. Diffuse, less prominent fibrin(ogen) extravasation was seen in the subcortical white matter of one iNPH individual. Increasing degree of fibrinogen extravasation in cerebral cortex was significantly associated with increasing degree of astrogliosis and with reduced expression of perivascular AQP4 and Dp71. ConclusionsThe present results provide evidence of BBB dysfunction in iNPH. The BBB leakage of blood proteins may render for impaired neurovascular units in iNPH patients.

Blood-Brain Barrier Dysfunction in Idiopathic Intracranial Hypertension.

Idiopathic intracranial hypertension (IIH) is traditionally considered benign and characterized by symptoms related to increased intracranial pressure, including headache and impaired vision. We have previously demonstrated that brains of IIH patients exhibit patchy astrogliosis, increased perivascular expression of the water channel aquaporin-4 (AQP4) as well as degenerating pericyte processes and capillary basement membranes. Given the established association between pericyte degeneration and blood-brain barrier (BBB) dysfunction, we investigated blood protein leakage by light microscopic immunohistochemistry. We also assessed perivascular AQP4 expression by immunogold transmission electron microscopy. The study included 14 IIH patients and 14 reference (REF) subjects undergoing neurosurgery for epilepsy, aneurysm, or tumor. Evidence of BBB dysfunction, measured as area extravasated fibrinogen/fibrin, was significantly more pronounced in IIH than REF individuals. The extent of extravasated fibrinogen was positively correlated with increasing degree of astrogliosis and vascular AQP4 immunoreactivity, determined by light microscopy. Immunogold transmission electron microscopy revealed no overall changes in AQP4 expression at astrocytic vascular endfeet in IIH (n = 8) compared to REF (n = 11) individuals. Our results provide evidence of BBB leakage in IIH, signifying that IIH is a more serious neurodegenerative disease than previously considered.

Differential expression of the c-fos protein and synaptophysin in zebrin II positive and zebrin II negative cerebellar cortical areas in 4-aminopyridine seizures

The present study examined temporal activation patterns of rat cerebellar cortical neurons in 4-aminopyridine induced seizures, using c-fos protein as a marker of neuronal activity. C-fos-containing cells were counted in each cerebellar cortical layer, and cell count was compared between zebrin II positive and zebrin II negative bands of the lobules of the vermis and cerebellar hemispheres. We found significant activation of granule cells and interneurons of the molecular layer in zebrin II positive bands. The Purkinje cells, in contrast, exhibited non-significant, scattered c-fos immunoreactivity across all bands. Fluctuation of synaptophysin expression in the mossy fibre rosettes of the granular layer was determined via light microscopic immunohistochemistry. We detected a transient, significant decrease in synaptophysin staining density following 4-aminopyridine seizures, which may indicate short-term synaptic depression. We also identified different timing of increased c-fos expression in the neurons of the cerebellar cortex in different cortical zones. In particular, the activation pattern of the interneurons of the molecular layer reflected the climbing fibre distribution, reflecting the zonal olivo-cortico-nuclear organization. Seizure-induced activation of the granule cells corresponded with the zebrin II positive zones. This observation raises the possibility that zebrin II positive compartments may be more susceptible to cerebellar convulsions.

Subcellular organization of UBE3A in human cerebral cortex

BackgroundLoss of UBE3A causes Angelman syndrome, whereas excess UBE3A activity appears to increase the risk for autism. Despite this powerful association with neurodevelopmental disorders, there is still much to be learned about UBE3A, including its cellular and subcellular organization in the human brain. The issue is important, since UBE3A’s localization is integral to its function.MethodsWe used light and electron microscopic immunohistochemistry to study the cellular and subcellular distribution of UBE3A in the adult human cerebral cortex. Experiments were performed on multiple tissue sources, but our results focused on optimally preserved material, using surgically resected human temporal cortex of high ultrastructural quality from nine individuals.ResultsWe demonstrate that UBE3A is expressed in both glutamatergic and GABAergic neurons, and to a lesser extent in glial cells. We find that UBE3A in neurons has a non-uniform subcellular distribution. In somata, UBE3A preferentially concentrates in euchromatin-rich domains within the nucleus. Electron microscopy reveals that labeling concentrates in the head and neck of dendritic spines and is excluded from the PSD. Strongest labeling within the neuropil was found in axon terminals.ConclusionsBy highlighting the subcellular compartments in which UBE3A is likely to function in the human neocortex, our data provide insight into the diverse functional capacities of this E3 ligase. These anatomical data may help to elucidate the role of UBE3A in Angelman syndrome and autism spectrum disorder.

Cell differentiation in cardiac myxomas: confocal microscopy and gene expression analysis after laser capture microdissection.

Cardiac myxomas are rare tumors with a heterogeneous cell population including properly neoplastic (lepidic), endothelial and smooth muscle cells. The assessment of neoplastic (lepidic) cell differentiation pattern is rather difficult using conventional light microscopy immunohistochemistry and/or whole tissue extracts for mRNA analyses. In a preliminary study, we investigated 20 formalin-fixed and paraffin-embedded cardiac myxomas by means of conventional immunohistochemistry; in 10/20 cases, cell differentiation was also analyzed by real-time RT-PCR after laser capture microdissection of the neoplastic cells, whereas calretinin and endothelial antigen CD31 immunoreactivity was localized in 4/10 cases by double immunofluorescence confocal microscopy. Gene expression analyses of α-smooth muscle actin, endothelial CD31 antigen, alpha-cardiac actin, matrix metalloprotease-2 (MMP2) and tissue inhibitor of matrix metalloprotease-1 (TIMP1) was performed on cDNA obtained from either microdissected neoplastic cells or whole tumor sections. We found very little or absent CD31 and α-Smooth Muscle Actin expression in the microdissected cells as compared to the whole tumors, whereas TIMP1 and MMP2 genes were highly expressed in both ones, greater levels being found in patients with embolic phenomena. α-Cardiac Actin was not detected. Confocal microscopy disclosed two different signals corresponding to calretinin-positive myxoma cells and to endothelial CD31-positive cells, respectively. In conclusion, the neoplastic (lepidic) cells showed a distinct gene expression pattern and no consistent overlapping with endothelial and smooth muscle cells or cardiac myocytes; the expression of TIMP1 and MMP2 might be related to clinical presentation; larger series studies using also systematic transcriptome analysis might be useful to confirm the present results.

Expression of vesicular glutamate transporters in transient receptor potential ankyrin 1 (TRPA1)-positive neurons in the rat trigeminal ganglion

Transient receptor potential ankyrin 1 (TRPA1), a cold receptor in sensory neurons activated by a variety of stimuli, is implicated in nociception and mechanotransduction. To help understand the vesicular glutamate transporter (VGLUT)-mediated glutamate signaling in TRPA1-immunopositive (+) neurons, we examined the expression of VGLUT1 and VGLUT2 in the TRPA1+ neurons in the male rat trigeminal ganglion (n = 19) under normal conditions and following experimental inflammation in the vibrissal pad by light microscopic immunohistochemistry (n = 11), western blot (n = 8), and quantitative analysis.One half (50.8%, 250/492) of the TRPA1+ neurons expressed VGLUT2, and a small fraction (8.3%, 57/683) also expressed VGLUT1. The majority of the VGLUT2-expressing TRPA1+ (VGLUT2+/TRPA1+) neurons coexpressed the markers of peptidergic and non-peptidergic neurons, CGRP, IB4, and TRPV1 but not the markers of neurons with myelinated fibers, NF200 and parvalbumin. In contrast, most VGLUT1+/TRPA1+ neurons coexpressed NF200 and parvalbumin but rarely expressed CGRP, IB4, or TRPV1. Following experimental inflammation, the fraction of VGLUT2+ (experimental vs. control: 34.7% vs. 22.3%), TRPA1+ (39.3% vs. 25.3%), and VGLUT2+/TRPA1+ (60.7% vs. 49.7%) neurons and the protein levels for TRPA1 and VGLUT2 increased significantly, compared to control, whereas the fraction of VGLUT1+ and VGLUT1+/TRPA1+ neurons and the protein level for VGLUT1 remained unchanged.These findings suggest that both VGLUT1 and VGLUT2 are involved in the glutamate signaling in TRPA1+ neurons under normal conditions in the male rats, and raise a possibility that VGLUT2 may play a role in the TRPA1-induced hypersensitivity following inflammation.

Molecular mechanisms underlying histological and biochemical changes induced by nitrate in rat liver and the efficacy of S-Allylcysteine

ABSTRACTThis study was designed to investigate structural and ultra-structural changes induced by nitrate in rat liver, examine their molecular basis, and evaluate the possible protective role of S-Allylcysteine (SAC). Adult male albino rats were assigned to: control, SAC, nitrate, and nitrate+ SAC groups. Serum ALT and AST were measured. Liver samples were processed for light and electron microscope examinations, biochemical analysis, real-time PCR, and immunohistochemistry of heat shock protein 70 (HSP70) and Bcl-2. Nitrate induced histopathological and biochemical alterations in rat liver. The underlying mechanisms included nitrosative and oxidative stress, inflammation, fibrosis, and apoptosis that are alleviated by SAC.

A developmental study of glutamatergic neuron populations in the ventrobasal and the lateral geniculate nucleus of the thalamus: Comparing Genetic Absence Rats from Strasbourg (GAERS) and normal control wistar rats

An imbalance of GABAergic inhibition and glutamatergic excitation is suspected to be the cause of absence epileptic seizures. Absence seizures are known to be generated in thalamocortical circuitry. In the present study we used light microscopy immunohistochemistry to quantify the density of glutamate+ve neurons at two developmental stages (P10 and P60) in two thalamic nuclei, the ventrobasal (VB) and lateral geniculate nucleus (LGN) in Wistar rats and compared the results with similar data obtained from genetic absence epilepsy rats from Strasbourg (GAERS).Rats were perfused transcardially with glutaraldehyde and paraformaldehyde fixative, then samples from VB and LGN were removed from each animal and sectioned. The glutamatergic neurons were labelled using light-microscopic glutamate immunohistochemistry. The disector method was used to quantify the glutamate+ve neurons in VB and LGN of GAERS and Wistar rats. The data were statistically analyzed.The distribution of the glutamate+ve neurons in the VB thalamic nucleus showed a significant reduction in the neuronal profiles per unit thalamic area from P10 to P60 in both Wistar and GAERS. The decrease was greater in the GAERS compared to the Wistar animals. However, in the LGN no reduction was observed either in the Wistar or in the GAERS. Comparing the density of glutamate+ve neurons in the VB thalamic nucleus of P10 of Wistar animals with of P10 GAERS showed statistically significant greater densities of these neurons in GAERS than in the Wistar rats. However no significant difference was present at P60 between the Wistar and GAERS animals.The disproportional decrease in GAERS may be related to the onset of absence seizures or may be related to neurogenesis of absence epilepsy.

Corticotropin-releasing hormone (CRH)-immunoreactive (IR) axon varicosities target a subset of growth hormone-releasing hormone (GHRH)-IR neurons in the human hypothalamus

It is a general consensus that stress is one of the major factors that suppresses growth. Previous studies revealed that the catecholaminergic and neuropeptide Y (NPY) systems, involved in the activation of stress-related neuronal circuits, influence growth hormone (GH)-release via modulating growth hormone-releasing hormone (GHRH) secretion. Indeed, catecholaminergic and NPY-immunoreactive (IR) axon varicosities abut on the surface of the GHRH neurons forming contacts. These juxtapositions appear to be real synapses and may represent the morphological substrate of the impact of stress on growth. In addition to catecholamines and NPY, there is a vast amount of evidence that corticotropin-releasing hormone (CRH), a major stress hormone, also influences GH secretion. Whether this modulatory effect is direct, or indirect, via the hypothalamic GHRH system, has not been elucidated yet.In the present study, we examined the possibility that CRH influences GH secretion via modulating the GHRH release by direct synaptic mechanisms. Since the verification of these synapses by electron microscopy is problematic in human due to the long post mortem time, in order to reveal the putative CRH-GHRH juxtapositions, light microscopic double label immunohistochemistry was utilized. In the infundibular nucleus, a subset (6%) of the GHRH perikarya received abutting CRH fiber varicosities forming multiple contacts while passing by. No gaps appeared between the contacting elements. The morphology of these CRH-GHRH juxtapositions suggests that, among other neurotransmitters/neuromodulators, CRH influences growth by modulating the hypothalamic GHRH secretion via direct synaptic mechanisms.

Evaluation, Diagnosis, and Staging of Cutaneous Lymphoma.

Primary cutaneous lymphomas (PCLs) are an extremely heterogeneous group of non-Hodgkin lymphomas that manifest in the skin. Their diagnosis is complex and based on clinical lesion type and evaluation of findings on light microscopic examination, immunohistochemistry and molecular analysis of representative skin biopsies. The evaluation, classification, and staging system is unique for mycosis fungoides (MF) and Sézary syndrome (SS), the most common subtypes of cutaneous T-cell lymphoma (CTCL) versus the other subtypes of Non-MF/Non-SS CTCL and the subtypes of cutaneous B-cell lymphoma (CBCL). Since current treatment is stage-based, it is particularly important that the correct diagnosis and stage be ascertained initially. The purpose of this article is to review the current evaluation, diagnosis, classification, staging, assessment techniques, and response criteria for the various types of both T-cell and B-cell PCLs.

Expression of vesicular glutamate transporters in transient receptor potential melastatin 8 (TRPM8)-positive dental afferents in the mouse

Transient receptor potential melastatin 8 (TRPM8) is activated by innocuous cool and noxious cold and plays a crucial role in cold-induced acute pain and pain hypersensitivity. To help understand the mechanism of TRPM8-mediated cold perception under normal and pathologic conditions, we used light microscopic immunohistochemistry and Western blot analysis in mice expressing a genetically encoded axonal tracer in TRPM8-positive (+) neurons. We investigated the coexpression of TRPM8 and vesicular glutamate transporter 1 (VGLUT1) and VGLUT2 in the trigeminal ganglion (TG) and the dental pulp before and after inducing pulpal inflammation.Many TRPM8+ neurons in the TG and axons in the dental pulp expressed VGLUT2, while none expressed VGLUT1. TRPM8+ axons were dense in the pulp horn and peripheral pulp and also frequently observed in the dentinal tubules. Following pulpal inflammation, the proportion of VGLUT2+ and of VGLUT2+/TRPM8+ neurons increased significantly, whereas that of TRPM8+ neurons remained unchanged.Our findings suggest the existence of VGLUT2 (but not VGLUT1)-mediated glutamate signaling in TRPM8+ neurons possibly underlying the cold-induced acute pain and hypersensitivity to cold following pulpal inflammation.

Observations on the expression of human papillomavirus major capsid protein in HeLa cells.

BackgroundThe goal of this study was to identify the nature of the inclusion bodies that have been found in HeLa cells (cervical cancer immortal cell line) by electron microscope and to determine whether the major capsid protein (L1) of human papillomavirus (HPV) can be expressed in HPV-positive uterine cervix cancer cells.MethodsHPV L1 protein expression in HeLa cells was detected with anti-HPV L1 multivalent mice monoclonal antibody and rabbit polyclonal anti-HPV L1 antibody by ELISA, light microscope immunohistochemistry, electron microscope immunocytochemistry and Western blotting assays. Reverse transcriptional PCR (RT-PCR) was performed to detect the transcription of L1 mRNA in HeLa cells. The immortalized human keratinocyte HeCat was used as the negative control.ResultsHPV L1 proteins reacted positively in the lysate of HeLa cells by ELISA assays. HRP labeled light microscope immunohistochemistry assay showed that there was a strong HPV L1 positive reaction in HeLa cells. Under the electron microscope, irregular shaped inclusion bodies, assembled by many small and uniform granules, had been observed in the cytoplasm of some HeLa cells. These granules could be labeled by the colloidal gold carried by HPV L1 antibody. The Western blotting assay showed that there was a L1 reaction strap at 80–85 kDa in the HeLa cell lysates, hence demonstrating the existence of HPV18 L1 in HeLa cells. RT-PCR assay showed that the L1 mRNA was transcribed in HeLa cells.ConclusionsThe inclusion bodies found in the cytoplasm of HeLa cells are composed of HPV18 L1 protein. Since HeLa cell line is a type of cervical cancer cells, this implies that HeLa cells have the ability to express HPV L1 proteins.