Injections Of Horseradish Peroxidase Research Articles

Progressive human-like tauopathy with downstream neurodegeneration and neurovascular compromise in a transgenic rat model

Tauopathies, including frontotemporal dementia (FTD) and Alzheimer’s disease (AD), clinically present with progressive cognitive decline and the deposition of neurofibrillary tangles (NFTs) in the brain. Neurovascular compromise is also prevalent in AD and FTD however the relationship between tau and the neurovascular unit is less understood relative to other degenerative phenotypes. Current animal models confer the ability to recapitulate aspects of the CNS tauopathies, however, existing models either display overaggressive phenotypes, or do not develop neuronal loss or genuine neurofibrillary lesions. In this report, we communicate the longitudinal characterization of brain tauopathy in a novel transgenic rat model, coded McGill-R955-hTau. The model expresses the longest isoform of human P301S tau. Homozygous R955-hTau rats displayed a robust, progressive accumulation of mutated human tau leading to the detection of tau hyperphosphorylation and cognitive deficits accelerating from 14 months of age. This model features extensive tau hyperphosphorylation with endogenous tau recruitment, authentic neurofibrillary lesions, and tau-associated neuronal loss, ventricular dilation, decreased brain volume, and gliosis in aged rats. Further, we demonstrate how neurovascular integrity becomes compromised at aged life stages using a combination of electron microscopy, injection of the tracer horseradish peroxidase and immunohistochemical approaches.

Intraneural Topography of Rat Sciatic Axons: Implications for Polyethylene Glycol Fusion Peripheral Nerve Repair.

Peripheral nerve injuries are the most common type of nerve trauma. We have been working with a novel repair technique using a plasmalemmal fusogen, polyethylene glycol (PEG), to re-fuse the membranes of severed axons. PEG-fusion repair allows for immediate re-innervation of distal targets, prevents axonal degeneration, and improves behavioral recovery. PEG-fusion of severed axons is non-specific, and we have previously reported that following injury and PEG-fusion misconnections between spinal motoneurons and their distal targets were present. Surprisingly, appropriately paired proximal and distal motor axons were observed in all PEG-fused animals. We hypothesized that a topographic organization of axons contributing to the sciatic nerve could explain the incidence of appropriate connections. We traced the course of specific axon populations contributing to the sciatic nerve in young adult male and female rats. Following intraneural injection of Fast Blue into the tibial branch, labeled axons were confined to a discrete location throughout the course of the nerve. Following intramuscular injection of cholera toxin-conjugated horseradish peroxidase into the anterior tibialis, labeled axons were confined to a smaller but still discrete location throughout the nerve. In both cases, the relative locations of labeled axons were consistent bilaterally within animals, as well as across animals and sexes. Thus, the relatively consistent location of specific axon populations could allow for realignment of appropriate populations of axons, and enhanced behavioral recovery seen in PEG-fused animals. Knowing the organization of axons within the sciatic nerve promotes accurate territory realignment during repair, therefore aiding in recovery outcomes.

Blockade of retinal or cortical activity does not prevent the development of callosal patches normally associated with ocular dominance columns in primary visual cortex.

Callosal patches in primary visual cortex of Long Evans rats, normally associated with ocular dominance columns, emerge by postnatal day 10 (P10), but they do not form in rats monocularly enucleated a few days before P10. We investigated whether we could replicate the results of monocular enucleation by using tetrodotoxin (TTX) to block neural activity in one eye, or in primary visual cortex. Animals received daily intravitreal (P6-P9) or intracortical (P7-P9) injections of TTX, and our physiological evaluation of the efficacy of these injections indicated that the blockade induced by a single injection lasted at least 24h. Four weeks later, the patterns of callosal connections in one hemisphere were revealed after multiple injections of horseradish peroxidase in the other hemisphere. We found that in rats receiving either intravitreal or cortical injections of TTX, the patterns of callosal patches analyzed in tangential sections from the flattened cortex were not significantly different from the pattern in normal rats. Our findings, therefore, suggest that the effects of monocular enucleation on the distribution of callosal connections are not due to the resulting imbalance of afferent ganglion cell activity, and that factors other than neural activity are likely involved.

Effects of different nerve autografts on greater auricular nerve deficit in rabbits

Effects of different nerve autografts on greater auricular nerve deficit in rabbits

Annals of Neurology: Volume 84, Number 3, September 2018

Annals of NeurologyVolume 84, Issue 3 Cover ImageFree Access Annals of Neurology: Volume 84, Number 3, September 2018 First published: 24 September 2018 https://doi.org/10.1002/ana.25336AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract An electron micrograph of a blood vessel in the cerebral cortex of a mouse 6 hours after cortical spreading depression. The animal received an intravenous injection of horseradish peroxidase (dark) which fills the blood vessel. Transudation of fluid from the lumen of the vessel occurs via vesicular transport (small black vesicles making their way from the lumen to the surface), not by opening of endothelial tight junctions. See Sadeghian, et al., pages 409–423. Ann Neurol 2018;84:1–1 Volume84, Issue3September 2018 RelatedInformation

Neurons of visual thalamic nuclei projecting to telencephalon express different types of calcium-binding proteins: A combined immunocytochemical and tracer study

In turtles (Testudo horsfieldi, Emys orbicularis), immunoreactivity to calbindin (CB), parvalbumin (PV), calretinin (CR) and co-localization of CB and PV were studied in neurons of the visual thalamic nuclei (Rot, GLd) projecting to the telencephalon using a combination of immunohistochemical and tracer methods. The prevalence of CB-immunoreactive (-ir) neurons in Rot, CB-ir and CR-ir neurons in GLd, and a smaller number of PV-ir neurons in both nuclei was shown. Double immunofluorescent labeling revealed that within both nuclei PV and CB are colocalized in most PV-ir and fewer CB-ir neurons. After injection of horseradish peroxidase into the Rot and GLs telencephalic projection fields, retrograde labeling was found in corresponding thalamic projection neurons immunoreactive to all the three proteins. After introduction of the fluorescent tracer Fluo-gold into the same telencephalic regions, retrograde labeling was detected in Rot and GLd neurons immunoreactive only to PV and CB as well as in neurons with colocalization of both proteins. These findings provide further evidence that in turtles the CB component prevails in the rotundo-telencephalic pathway while the CB/CR component is dominant in the geniculotelencephalic pathway. The role of functional specialization in segregation of neurons expressing distinct types of calcium-binding proteins is postulated.

Metabotropic glutamate receptor 5 shows different patterns of localization within the parallel visual pathways in macaque and squirrel monkeys.

Glutamate is used as an excitatory neurotransmitter by the koniocellular (K), magnocellular (M), and parvocellular (P) pathways to transfer signals from the primate lateral geniculate nucleus (LGN) to primary visual cortex (V1). Glutamate acts through both fast ionotropic receptors, which appear to carry the main sensory message, and slower, modulatory metabotropic receptors (mGluRs). In this study, we asked whether mGluR5 relates in distinct ways to the K, M, and P LGN axons in V1. To answer this question, we used light microscopic immunocytochemistry and preembedding electron microscopic immunogold labeling to determine the localization of mGluR5 within the layers of V1 in relation to the K, M, and P pathways in macaque and squirrel monkeys. These pathways were labeled separately via wheat germ agglutinin–horseradish peroxidase (WGA–HRP) injections targeting the LGN layers. mGluR5 is of interest because it: 1) has been shown to be expressed in the thalamic input layers; 2) appears to be responsible for some types of oscillatory firing, which could be important in the binding of visual features; and 3) has been associated with a number of sensory-motor gating-related pathologies, including schizophrenia and autism. Our results demonstrated the presence of mGluR5 in the neuropil of all V1 layers. This protein was lowest in IVCα (M input) and the infragranular layers. In layer IVC, mGluR5 also was found postsynaptic to about 30% of labeled axons, but the distribution was uneven, such that postsynaptic mGluR5 label tended to occur opposite smaller (presumed P), and not larger (presumed M) axon terminals. Only in the K pathway in layer IIIB, however, was mGluR5 always found in the axon terminals themselves. The presence of mGluR5 in K axons and not in M and P axons, and the presence of mGluR5 postsynaptic mainly to smaller P and not larger M axons suggest that the response to the release of glutamate is modulated in distinct ways within and between the parallel visual pathways of primates.

Central pupillary light reflex circuits in the cat: I. The olivary pretectal nucleus.

The central pathways subserving the feline pupillary light reflex were examined by defining retinal input to the olivary pretectal nucleus (OPt), the midbrain projections of this nucleus, and the premotor neurons within it. Unilateral intravitreal wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) injections revealed differences in the pattern of retinal OPt termination on the two sides. Injections of WGA-HRP into OPt labeled terminals bilaterally in the anteromedian nucleus, and to a lesser extent in the supraoculomotor area, centrally projecting Edinger-Westphal nucleus, and nucleus of the posterior commissure. Labeled terminals, as well as retrogradely labeled multipolar cells, were present in the contralateral OPt, indicating a commissural pathway. Injections of WGA-HRP into the anteromedian nucleus labeled fusiform premotor neurons within the OPt, as well as multipolar cells in the nucleus of the posterior commissure. Connections between retinal terminals and the pretectal premotor neurons were characterized by combining vitreous chamber and anteromedian nucleus injections of WGA-HRP in the same animal. Fusiform-shaped, retrogradely labeled cells fell within the anterogradely labeled retinal terminal field in the OPt. Ultrastructural analysis revealed labeled retinal terminals containing clear spherical vesicles. They contacted labeled pretectal premotor neurons via asymmetric synaptic densities. These results provide an anatomical substrate for the pupillary light reflex in the cat. Pretectal premotor neurons receive direct retinal input via synapses suggestive of an excitatory drive, and project directly to nuclei containing preganglionic motoneurons. These projections are concentrated in the anteromedian nucleus, indicating its involvement in the pupillary light reflex.

SHP-1 regulates neuroinvasion of neurotropic virus into the CNS (VIR5P.1035)

Abstract Viral infections in the central nervous system (CNS) may have devastating clinical consequences to the host. Neurotropic virus infections often begin in peripheral tissues, which then spread to the CNS via peripheral nerves following entry into nerve terminals by receptor-mediated endocytosis and subsequent retrograde axonal transport into the CNS compartment. In our lab, a possible novel role for the protein tyrosine phosphatase, SHP-1, in regulating neuroinvasion via peripheral nerves of a neurotropic virus (Theiler’s Murine Encephalomyelitis Virus, TMEV) is being investigated. Preliminary data using immunohistochemistry of TMEV showed extensive staining of spinal cord neurons after intraperitoneal inoculation in mice with a null mutation in the SHP-1 gene (me/me mice) compared to wild type mice. Further, intramuscular (IM) inoculations of TMEV caused virus infection in spinal cord neurons and subsequent paralysis of SHP-1-deficient mice but not of wild type mice. IM and intraperitoneal injections (IP) of retrograde axonal transport tracers fluorogold and horseradish peroxidase showed increased tracer levels in the spinal cords of SHP-1-deficient mice compared to wild type mice. Therefore, we propose SHP-1 may play a role in regulating viral neuroinvasion by controlling retrograde axonal transport from peripheral sites of infection. In this sense, SHP-1 may play novel roles in antiviral responses including inhibition of virus invasion and spreading in the CNS.

Whisker-related circuitry in the trigeminal nucleus principalis: Ultrastructure

Trigeminal (V) nucleus principalis (PrV) is the requisite brainstem nucleus in the whisker-to-barrel cortex model system that is widely used to reveal mechanisms of map formation and information processing. Yet, little is known of the actual PrV circuitry. In the ventral “barrelette” portion of the adult mouse PrV, relationships between V primary afferent terminals, thalamic-projecting PrV neurons, and gamma-aminobutyric acid (GABA)-ergic terminals were analyzed in the electron microscope. Primary afferents, thalamic-projecting cells, and GABAergic terminals were labeled, respectively, by Neurobiotin injections in the V ganglion, horseradish peroxidase injections in the thalamus, and postembedding immunogold histochemistry. Primary afferent terminals (Neurobiotin- and glutamate-immunoreactive) display asymmetric and multiple synapses predominantly upon the distal dendrites and spines of PrV cells that project to the thalamus. Primary afferents also synapse upon GABAergic terminals. GABAergic terminals display symmetric synapses onto primary afferent terminals, the somata and dendrites (distal, mostly) of thalamic-projecting neurons, and GABAergic dendrites. Thus, primary afferent inputs through the PrV are subject to pre- and postsynaptic GABAergic influences. As such, circuitry exists in PrV “barrelettes” for primary afferents to directly activate thalamic-projecting and inhibitory local circuit cells. The latter are synaptically associated with themselves, the primary afferents, and with the thalamic-projecting neurons. Thus, whisker-related primary afferent inputs through PrV projection neurons are pre- and postsynaptically modulated by local circuits.

Immuno- and Enzyme-histochemistry of HRP for Demonstration of Blood Vessel Permeability in Mouse Thymic Tissues by "In Vivo Cryotechnique".

It is difficult to understand the in vivo permeability of thymic blood vessels, but “in vivo cryotechnique” (IVCT) is useful to capture dynamic blood flow conditions. We injected various concentrations of horseradish peroxidase (HRP) with or without quantum dots into anesthetized mice via left ventricles to examine architectures of thymic blood vessels and their permeability at different time intervals. At 30 sec after HRP (100 mg/ml) injection, enzyme reaction products were weakly detected in interstitium around some thick blood vessels of corticomedullary boundary areas, but within capillaries of cortical areas. At 1 and 3 min, they were more widely detected in interstitium around all thick blood vessels of the boundary areas. At 10 min, they were diffusely detected throughout interstitium of cortical areas, and more densely seen in medullary areas. At 15 min, however, they were uniformly detected throughout interstitium outside blood vessels. At 30 min, phagocytosis of HRP by macrophages was scattered throughout the interstitium, which was accompanied by decrease of HRP reaction intensity in interstitial matrices. Thus, time-dependent HRP distributions in living mice indicate that molecular permeability and diffusion depend on different areas of thymic tissues, resulting from topographic variations of local interstitial flow starting from corticomedullary areas.

Effects of Strabismus and Monocular Deprivation on the Sizes of Callosal Cells in Cortical Fields 17 and 18 in the Cat Brain

Structural changes in the visual cortex were studied in conditions of deranged binocular experience by assessing the sizes (body areas) of callosal cells in fields 17 and 18 in monocularly deprived cats and in cats with convergent strabismus. These cells were detected by injection of horseradish peroxidase into columns in cortical fields 17 and 18 and the fields 17/18 transitional zone. In both groups, the mean size of callosal cells in field 17 was greater than normal, though this difference in field 18 was seen only in monocularly deprived cats. Differences in the mean sizes of field 17 and 18 cells in cats of the study groups were found to be due to the number of large cells. In cats with strabismus, callosal cells of size greater than 200 μm2 accounted for 58% of cells in field 17 and 8% in field 18. In monocularly deprived cats, there was no difference in the proportions of large callosal cells in these fields (28% and 26%, respectively). These data provide evidence that cytoarchitectonic changes occurred in layers of the visual cortex, serving as sources of interhemisphere connections, in conditions of early derangement of binocular experience.

Endolymphatic Periductal Channels: Possible Role in Ménière's Disease

Objectives: Characterize the osseous periductal channels surrounding the human endolymphatic duct. Endolymphatic hydrops is the fundamental pathophysiology underlying Ménière’s disease. Elucidating the regulation of endolymphatic circulation is central to understanding the cause of Ménière’s disease attacks of vertigo and hearing loss. Methods: Formalin-fixed, celloidin-embedded human temporal bones were sectioned to 20 microns, and every section was stained with hematoxylin & eosin. Amira software was used to construct a three dimensional image of the periductal channels. We performed immunohistochemical analysis using antibodies directed against vascular, lymphatic, and neural crest markers. The microanatomy was investigated with electron microscopy of the periductal channel epithelium. Finally, we injected horseradish peroxidase into the scala media of guinea pigs to investigate the flow patterns involving these peri-ductal channels. Results: Surrounding the human endolymphatic duct is a group of small channels that originate in the proximal cisternal area of the endolymphatic sac and terminate in the supporting tissue of the saccule, as demonstrated by 3D reconstructions. Endolymphatic hydrops was found in eight bones with blocked ducts and blocked or absent periductal channels. Proliferation of channels was evident in temporal bones when the channels were disrupted on one side. Immunohistochemical analysis results suggest a neuroectodermal origin, similar to the cells of the cochlear spiral ligament. Some contain micro-vessels with single endothelial walls that could represent lymphatics or empty capillaries while others suggest a complex absortive epithelium. Conclusions: This temporal bone study suggests a role for the periductal channels in absorption of endolymph.

Localization of masticatory motoneurons in the trigeminal motor nucleus of shrew and pig, with emphasis on the innervation ratio in the shrew

Objectives:We investigated the topographical representation of the masticatory muscles, primarily in the trigeminal motor nucleus (TMN) of the house musk shrew (Suncus murinus) and miniature pig, in a series of mammalian comparative anatomical studies. Additionally, correlations between motoneurons and muscles were investigated in order to examine the functionality of each muscle in the trigeminal motor system. Methods:Motoneurons were labeled using horseradish peroxidase (HRP) injection into each muscle innervated by the trigeminal nerve. In the shrew, the sizes and numbers of HRP-labeled neurons, muscle weight, and numbers of muscle fibers were measured in the innervated muscles, and correlation coefficients for the relationships among these parameters were calculated. Results:The motoneuron cluster of each muscle was arranged in the TMN in a manner similar to that observed in other previously reported animals, but the distribution of the lateral pterygoid motoneurons varied between species. In addition, considerably higher overlap was observed in each pig jaw-closer motoneuron cluster compared with other animals. The approximate innervation ratios were as follows: masseter, 337; temporal, 322; anterior digastric, 137; medial pterygoid, 110; lateral pterygoid, 79; mylohyoid, 42; tensor veli palatini, 42; transverse mandibular, 16; and tensor tympani muscles, 5. ConclusionsThe distribution pattern of the masticatory motoneurons was clearly observed in the shrew and pig TMN, and various correlations between the motoneuron and innervated muscles were determined.

Effects of prednisolone on the dystrophin-associated proteins in the blood–brain barrier and skeletal muscle of dystrophic mdx mice

The mdx mouse, the most widely used animal model of Duchenne muscular dystrophy (DMD), develops a seriously impaired blood–brain barrier (BBB). As glucocorticoids are used clinically to delay the progression of DMD, we evaluated the effects of chronic treatment with α-methyl-prednisolone (PDN) on the expression of structural proteins and markers in the brain and skeletal muscle of the mdx mouse. We analyzed the immunocytochemical and biochemical expression of four BBB markers, including endothelial ZO-1 and occludin, desmin in pericytes, and glial fibrillary acidic protein (GFAP) in glial cells, and the expression of the short dystrophin isoform Dp 71, the dystrophin-associated proteins (DAPs), and aquaporin-4 (AQP4) and α–β dystroglycan (DG) in the brain. We evaluated the BBB integrity of mdx and PDN-treated mdx mice by means of intravascular injection of horseradish peroxidase (HRP). The expression of DAPs was also assessed in gastrocnemius muscles and correlated with utrophin expression, and laminin content was measured in the muscle and brain. PDN treatment induced a significant increase in the mRNA and protein content of the BBB markers; a reduction in the phosphorylation of occludin in the brain and of AQP4/β DG in both tissues; an increase of Dp71 protein content; and an increase of both mRNA and protein levels of the AQP4/α–β DG complex. The latter was associated with enhanced laminin and utrophin in the muscle. The HRP assay demonstrated functional restoration of the BBB in the PDN-treated mdx mice. Specifically, mdx mice showed extensive perivascular labeling due to escape of the marker, while HRP was exclusively intravascular in the PDN-treated mice and the controls. These data illustrate for the first time that PDN reverses the BBB alterations in the mdx mouse and re-establishes the proper expression and phosphorylation of β-DG in both the BBB and skeletal muscle. Further, PDN partially protects against muscle damage. The reduction in AQP4 and occludin phosphorylation, coupled with their anchoring to glial and endothelial membranes in PDN-treated mice, suggests that the drug may target the glial and endothelial cells. Our results suggest a novel mechanism for PDN action on cerebral and muscular function, restoring the link between DAPs and the extracellular matrix, most likely through protein kinase inactivation.

Aquaporin-4 expression and blood–spinal cord barrier permeability in canalicular syringomyelia

Noncommunicating canalicular syringomyelia occurs in up to 65% of patients with Chiari malformation Type I. The pathogenesis of this type of syringomyelia is poorly understood and treatment is not always effective. Although it is generally thought that syringomyelia is simply an accumulation of CSF from the subarachnoid space, the pathogenesis is likely to be more complex and may involve cellular and molecular processes. Aquaporin-4 (AQP4) has been implicated in numerous CNS pathological conditions involving fluid accumulation, including spinal cord edema. There is evidence that AQP4 facilitates the removal of extracellular water following vasogenic edema. The aim of this study was to investigate AQP4 expression and the structural and functional integrity of the blood-spinal cord barrier (BSCB) in a model of noncommunicating canalicular syringomyelia. A kaolin-induced model of canalicular syringomyelia was used to investigate BSCB permeability and AQP4 expression in 27 adult male Sprague-Dawley rats. Control groups consisted of nonoperated, laminectomy-only, and saline-injected animals. The structural integrity of the BSCB was assessed using immunoreactivity to endothelial barrier antigen. Functional integrity of the BSCB was assessed by extravasation of systemically injected horseradish peroxidase (HRP) at 1, 3, 6, or 12 weeks after surgery. Immunofluorescence was used to assess AQP4 and glial fibrillary acidic protein (GFAP) expression at 12 weeks following syrinx induction. Extravasation of HRP was evident surrounding the central canal in 11 of 15 animals injected with kaolin, and in 2 of the 5 sham-injected animals. No disruption of the BSCB was observed in laminectomy-only controls. At 12 weeks the tracer leakage was widespread, occurring at every level rostral to the kaolin injection. At this time point there was a decrease in EBA expression in the gray matter surrounding the central canal from C-5 to C-7. Aquaporin-4 was expressed in gray- and white-matter astrocytes, predominantly at the glia limitans interna and externa, and to a lesser extent around neurons and blood vessels, in both control and syrinx animals. Expression of GFAP and APQ4 directly surrounding the central canal in kaolin-injected animals was variable and not significantly different from expression in controls. This study demonstrated a prolonged disruption of the BSCB directly surrounding the central canal in the experimental model of noncommunicating canalicular syringomyelia. The disruption was widespread at 12 weeks, when central canal dilation was most marked. Loss of integrity of the barrier with fluid entering the interstitial space of the spinal parenchyma may contribute to enlargement of the canal and progression of syringomyelia. Significant changes in AQP4 expression were not observed in this model of canalicular syringomyelia. Further investigation is needed to elucidate whether subtle changes in AQP4 expression occur in canalicular syringomyelia.

The Sizes of Cells Providing Interhemisphere and Intrahemisphere Connections in the Cat Visual Cortex after Lesioning of Binocular Vision

The sizes (body areas) of 658 retrograde labeled cells in layers 2/3 of cortical fields 17 and 18 in both hemispheres were measured in intact cats, monocularly deprived cats, and cats with bilateral convergent strabismus after injection of horseradish peroxidase into the eye dominance columns of these fields. The mean size of callosal cells in all groups of cats was significantly greater than the mean size of cells providing intrahemisphere connections. The mean sizes of callosal cells in monocularly deprived cats and cats with strabismus were significantly greater than the mean size of callosal cells in intact cats, by 26.6% and 20.2%, respectively. The mean size of intrahemisphere cells in monocularly deprived cats was not different from the mean sizes of interhemisphere cells in cats with strabismus and intact cats. These data lead to the conclusion that impairments to binocular vision lead to increases in the sizes of callosal cells in the visual cortex.

Retinal ganglion cell loss on APP/PS1 transgenic mice with Alzheimer's disease

To investigate the retinal nerve cell changes in number by tests on APP/PS1 double transgenic mice. Experimental study. Comparison was made between 10 female 11-month-old APP/PS1 double transgenic mice with Alzheimer's disease (model group) and 10 non-transgenic female mice of the same background and age (control group). Retinal ganglion cell (RGC) was retrograde labeled by injecting horse radish peroxidase (HRP) into the bilateral optic nerve layer of the superior colliculus of two groups. After 48 hours, all mice were perfused sacrificed. The brains were frozen, mode slices, stained with methyl-alcohol-Congo. The retina peeled from right eye was conducted HRP histochemistry reaction to count RGC by computerized image analyzer. The left eyes were treated with paraffin-embedded, mode slices, stained with hematoxylin and eosin (HE) to count the neuron numbers of each retina layers. The neuron numbers of each retina layer for control group and model group were statistically analyzed using t test for independent samples. The brains showed positive in the model group and the brains showed negative in the control group after stained with methyl-alcohol-Congo. By counting retinal ganglion cells traced with HRP, the number of RGC was statistical different between two groups at 112.78 ± 7.70 within per field for the control group against 78.72 ± 11.35 within per field for the model group. The RGC of the model group was comparatively much less than the control group (t = 27.28, P < 0.01). By counting cells of retina stained with HE, the number of inner and outer nuclear layer cells were not statistical different between two groups of each layer (P > 0.05), the number of RGC in the control group and the model group were respectively 8.17 ± 1.17 within per field, 5.17 ± 0.75 within per field. The cells in retinal ganglion layer in the model group were significantly reduced (t = 52.88, P < 0.01). The results imply that the number of RGC reduced in 11-month-old APP/PS1 double transgenic mice with Alzheimer's disease.

Protection of Blood Retinal Barrier and Systemic Vasculature by Insulin-Like Growth Factor Binding Protein-3

Previously, we showed that insulin growth factor (IGF)-1 binding protein-3 (IGFBP-3), independent of IGF-1, reduces pathological angiogenesis in a mouse model of the oxygen-induced retinopathy (OIR). The current study evaluates novel endothelium-dependent functions of IGFBP-3 including blood retinal barrier (BRB) integrity and vasorelaxation. To evaluate vascular barrier function, either plasmid expressing IGFBP-3 under the regulation of an endothelial-specific promoter or a control plasmid was injected into the vitreous humor of mouse pups (P1) and compared to the non-injected eyes of the same pups undergoing standard OIR protocol. Prior to sacrifice, the mice were given an injection of horseradish peroxidase (HRP). IGFBP-3 plasmid-injected eyes displayed near-normal vessel morphology and enhanced vascular barrier function. Further, in vitro IGFBP-3 protects retinal endothelial cells from VEGF-induced loss of junctional integrity by antagonizing the dissociation of the junctional complexes. To assess the vasodilatory effects of IGFBP-3, rat posterior cerebral arteries were examined in vitro. Intraluminal IGFBP-3 decreased both pressure- and serotonin-induced constrictions by stimulating nitric oxide (NO) release that were blocked by L-NAME or scavenger receptor-B1 neutralizing antibody (SRB1-Ab). Both wild-type and IGF-1-nonbinding mutant IGFBP-3 (IGFBP-3NB) stimulated eNOS activity/NO release to a similar extent in human microvascular endothelial cells (HMVECs). NO release was neither associated with an increase in intracellular calcium nor decreased by Ca2+/calmodulin-dependent protein kinase II (CamKII) blockade; however, dephosphorylation of eNOS-Thr495 was observed. Phosphatidylinositol 3-kinase (PI3K) activity and Akt-Ser473 phosphorylation were both increased by IGFBP-3 and selectively blocked by the SRB1-Ab or PI3K blocker LY294002. In conclusion, IGFBP-3 mediates protective effects on BRB integrity and mediates robust NO release to stimulate vasorelaxation via activation of SRB1. This response is IGF-1- and calcium-independent, but requires PI3K/Akt activation, suggesting that IGFBP-3 has novel protective effects on retinal and systemic vasculature and may be a therapeutic candidate for ocular complications such as diabetic retinopathy.

A Direct Hippocampo‐Cerebellar Projection in Chicken

Our previous studies suggested that a direct hippocampo-cerebellar projection might exist in the chicken. To confirm such a presumption of hippocampo-cerebellar interactions, horseradish peroxidase (HRP) was used as a retrograde tracer to be injected into the white matter of the folia VI-VIII of the cerebellum in young and adult chickens. In another set of experiments, young chickens were subjected to electrolytic lesions of the hippocampal formation (HF), especially the ventromedial portion, and the cerebellum was observed with the electron microscope to find neuronal degeneration in the HF. Following injections of HRP into the cerebellum, a large number of labeled neurons were found in the area APHm-APHim of the HF in the young and adult chickens. As a result of the electrolytic lesions of the APHm-APHim in the HF, many large degenerated nerve fibers were found in the white matter in the vicinity of the lateral nucleus of the cerebellum, and some small degenerated fibers were found in the white matter of the folia VI-VIII. In the cerebellar cortex of folia VI-VIII, degenerated axonal terminals occurred in both the molecular and Purkinje layers, but not in the granular layer. In the lateral nucleus, some dark degenerating axonal terminals were recognized to connect with the perikarya of neurons of this nucleus. The present experiments demonstrate that the APHm-APHim of the HF directly projected to the cortex of folia VI-VIII and the lateral nucleus of the cerebellum in young chickens.