Immunohistochemical identification and fiber type specific localization of protein kinase C isoforms in equine skeletal muscle

OBJECTIVE: To investigate whether protein kinase C (PKC) isoforms are expressed in equine skeletal muscle and determine their distribution in various types of fibers by use of immunofluorescence microscopy. ANIMALS: 5 healthy adult Dutch Warmblood horses. PROCEDURE: In each horse, 2 biopsy specimens were obtained from the vastus lateralis muscle. Cryosections of equine muscle were stained with PKC isoform (alpha, beta1, beta2, delta, epsilon, or zeta)-specific polyclonal antibodies and examined by use of a fluorescence microscope. Homogenized muscle samples were evaluated via western blot analysis. RESULTS: The PKC alpha, beta1, beta2, delta, epsilon, and zeta isoforms were localized within the fibers of equine skeletal muscle. In addition, PKC alpha and beta2 were detected near or in the plasma membrane of muscle cells. For some PKC isoforms, distribution was specific for fiber type. Staining of cell membranes for PKC alpha was observed predominantly in fibers that reacted positively with myosin heavy chain (MHC)-IIa; PKC delta and epsilon staining were more pronounced in MHC-I-positive fibers. In contrast, MHC-I negative fibers contained more PKC zeta than MHC-I-positive fibers. Distribution of PKC beta1 was equal among the different fiber types. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that PKC isoforms are expressed in equine skeletal muscle in a fiber type-specific manner. Therefore, the involvement of PKC isoforms in signal transduction in equine skeletal muscle might be dependent on fiber type.     

Immunohistochemical study of caveolin-1 and -2 in the rat retina

The expression of caveolin-1 and -2 in the retina was examined; Western blot analysis showed that both were present. Immunohistochemistry indicated that caveolin-1 was expressed in the majority of retinal layers, including the ganglion cell layer, inner plexiform layer, outer plexiform layer, and in the vascular endothelial cells of the retina. Caveolin-2 was primarily immunostained in the vessels, but in a few other elements as well. This is the first demonstration of caveolin differential expression in the retina of rats, and suggests that caveolin plays an important role in signal transduction in glial cells and neuronal cells.     

Participation of specific PKC isozymes in the inhibitory effect of ET-1 on progesterone accumulation in cells isolated from early- and mid-phase corpora lutea

Expression of PKC alpha, beta I, beta II, epsilon and micro has been demonstrated in the whole bovine CL with PKC epsilon being differentially expressed as a function of development. In experiment 1 we have investigated the amount of mRNA encoding PKC epsilon at different stages of luteal development (days 1, 4, 10 and 17). In experiment 2, the cellular source of luteal PKC isozymes was determined. Enriched steroidogenic (SC) and endothelial (EC) cells from day-10 CL were used to examine this question by Western blot analysis and immuno-histochemistry. In experiment 3, Western blot analysis was used to examine the ability of ET-1 to activate luteal PKC isozymes in day-10 CL. In experiment 4, the role of luteal PKC isozymes in the ET-1 mediated inhibition of P(4) accumulation in steroidogenic cell cultures from day-4 and day-10 CL was examined. Abundance of PKC epsilon mRNA gradually increased from day-1 to -10 with no further increase on day-17. In experiment 2, PKC epsilon was exclusively detected in SC (LLC and SLC). In contrast, PKC alpha, beta I and beta II were detected in both SC and EC, with EC expressing higher amounts of PKC isozymes. In day-10 CL, ET-1 induced cellular redistribution of PKC alpha, beta I, epsilon but not beta II. Inhibitors specific for conventional PKC isozymes as well as PKC epsilon were able to negate the inhibitory effects of ET-1 on P4 accumulation in the day 10 CL. In the day-4 CL, the inhibitory effect of ET-1 might be mediated via conventional PKC. Thus, an exclusive presence of PKC epsilon in luteal steroidogenic cells, its higher expression along with the ability of ET-1 to stimulate its activation in day-10 CL strongly suggests that this PKC isoform may play an important regulatory role in decreasing P(4) during luteal regression. Inhibition of P(4) by ET-1 in the early CL may be mediated via conventional PKC isozymes.     

Visual and quantitative electroencephalographic analysis of healthy young and adult cats under medetomidine sedation

A study was designed to investigate the effect of medetomidine sedation on quantitative electroencephalography (q-EEG) in healthy young and adult cats to determine objective guidelines for diagnostic EEG recordings and interpretation. Preliminary visual examination of EEG recordings revealed high-voltage low-frequency background activity. Spindles, k-complexes and vertex sharp transients characteristic of sleep or sedation were superimposed on a low background activity. Neither paroxysmal activity nor EEG burst-suppression were observed. The spectral analysis of q-EEG included four parameters, namely, relative power (%), and mean, median and peak frequency (Hz) of all four frequency bands (delta, theta, alpha and beta). The findings showed a prevalence of slow delta and theta rhythms as opposed to fast alpha and beta rhythms in both young (group A) and adult (group B) cats. A posterior gradient was reported for the theta band and an anterior gradient for the alpha and beta bands in both groups, respectively. The relative power value in group B compared to group A was significantly higher for theta, alpha and beta bands, and lower for the delta band. The mean and median frequency values in group B was significantly higher for delta, theta and beta bands and lower for the alpha band. The study has shown that a medetomidine sedation protocol for feline EEG may offer a method for investigating bio-electrical cortical activity. The use of q-EEG analysis showed a decrease in high frequency bands and increased activity of the low frequency band in healthy cats under medetomidine sedation.     

PKC isoenzymes in equine platelets and stimulus induced activation

Protein kinase C (PKC) is an important regulator of platelet activation and different isoenzymes can play positive and negative regulatory roles. The PKC isoenzymes expressed in equine platelets have not been documented but pharmacological inhibition has suggested a role for PKC delta (δ) in modulating responsiveness to platelet activating factor (PAF) (Brooks et al., 2009). Here the PKC isoenzyme profile in equine platelets has been characterised and PKCδ activation by PAF investigated. Platelet lysates were probed by Western blotting using a panel of antibodies against individual PKC isoenzymes. PKCδ and eight other isoenzymes were identified, namely classical PKCs alpha (α), beta (β), (both βI and βII) and gamma (γ), the novel PKCs epsilon (?), eta (η) and theta (θ) and atypical PKC zeta (ζ). Having shown PKCδ to be present, a method was developed to measure PAF-induced isoenzyme translocation by preparing cytosolic and membrane fractions from digitonin permeabilised platelets. Phorbol 12-myristate 13-acetate (PMA) was shown to cause translocation of PKCδ to the membrane within 5s. PAF also caused PKCδ translocation although the response occurred more slowly; a significant, 7.6 ± 1.2 fold, increase in band density compared to unstimulated platelets was observed at 15 min; p=0.036, n=3. These data support a role for PKCδ in regulating PAF-induced functional responses in equine platelets.     

Abnormal prion accumulation associated with retinal pathology in experimentally inoculated scrapie-affected sheep

The purpose of this study was to characterize the patterns of PrP(Sc) immunoreactivity in the retinae of scrapie-affected sheep and to determine the extent of retinal pathology as indicated by glial fibrillary acidic protein immunoreactivity (GFAP-IR) of Müller glia. Sections from the retina of 13 experimentally inoculated scrapie-affected and 2 negative control sheep were examined with immunohistochemical staining for PrP(Sc), GFAP, and PrP(Sc)/GFAP double staining. GFAP-IR of Müller glia is suggestive of retinal pathology in the absence of morphologic abnormality detected by light microscopy. Sheep with the least amount of PrP(Sc) in the retina have multifocal punctate aggregates of prion staining in the outer half of the inner plexiform layer and rarely in the outer plexiform layer. In these retinae, GFAP-IR is not localized with prion accumulation, but rather is present in moderate numbers of Müller glia throughout the sections of retina examined. The majority of sheep with retinal accumulation of PrP(Sc) have intense, diffuse PrP(Sc) staining in both plexiform layers, with immunoreactivity in the cytoplasm of multiple ganglion cells and lesser amounts in the optic fiber layer and between nuclei in nuclear layers. This intense PrP(Sc) immunoreactivity is associated with diffuse, intense GFAP-IR that extends from the inner limiting membrane to the outer limiting membrane. This is the first report of a prion disease in a natural host that describes the accumulation of PrP(Sc) in retina associated with retinal pathology in the absence of overt morphologic changes indicative of retinal degeneration.     

Development of the Retina in the Porcine Fetus A Light Microscopic Study

Morphogenesis of the porcine retina was studied using light microscopy from 4 weeks of gestation until birth (18 to 310 mm crown-rump length), and compared with the adult stage (6 months). Tissue samples were examined from the posterior and peripheral parts of the retina. At 18 mm the retina consists of an inner marginal layer and an outer layer of neuroblastic cells. At 18-40 mm the latter layer is divided into an inner and an outer neuroblastic layer by the transient layer of Chievitz. Subsequently, the development of the different retinal layers begins at the inner retinal border and moves progressively outwards; it also spreads from the posterior to the peripheral part of the neural retina. Many cells of the inner neuroblastic layer are prospective ganglionic cells which migrate inwards, thus forming the ganglion cell layer and the inner plexiform layer at 90 mm. At 120 mm, primitive horizontal cells appear within the outer neuroblastic layer. Separation of this layer into the inner nuclear, outer plexiform and outer nuclear layers is first evident at 180 mm. At this stage all retinal layers are present, except the layer of the photoreceptor cells which is not widespread until at 220 mm. The inner and outer segments of the photoreceptor cells lengthen considerably during the last month of gestation. During the late fetal stage the nerve fiber layer, the inner and outer plexiform layers and the layer of rods and cones all continue to increase in thickness. Concurrently, the ganglion cell layer and the inner and outer nuclear layers have reached their maximal thickness and become thinner. After the total thickness of the neural retina amounts to approximately 180 microns at two to three weeks before birth, it then thins to approximately 160 microns in the adult stage.     

Genomic sequencing of the bovine T cell receptor beta locus

The T cell, which plays an integral role in the coordination of the immune system, has a heterodimer receptor (TCR) that can exist in one of the two forms: alpha/beta or gamma/delta. Cells displaying the gamma/delta TCR comprise less than 5% of T cell populations in humans and mice. In the bovine system, however, gamma/delta populations can reach as high as 60%. Differences in T cell populations make the bovine system an excellent candidate for genomic TCR sequencing and multi-species comparisons.In an effort to characterize the bovine TCR loci, a genomic library was screened for the beta TCR gene. A shotgun sequencing library was constructed and preliminary analysis demonstrates that the organization of the bovine TCR beta constant regions is different from both humans and mice. The bovine beta locus appears to have a third constant region. Overall, the genomic characterization of the bovine TCR genes will provide insight into the evolution of T cell receptor.     

Pathogenetic studies of infection of the bovine fetus with bovine viral diarrhea virus. II. Ocular lesions.

Twenty-three susceptible pregnant heifers were inoculated with bovine viral diarrhea virus at 150 +/- 1 days of gestation. Seven additional heifers were inoculated between 65 and 115 days of gestation. Acute ocular lesions were seen in fetuses taken 17-21 days after inoculation of the dams at 150 days. By the fourth week, the acute lesions were beginning to resolve, and in newborn animals focal to total retinal atrophy was seen. The acute lesions were characterized by a mild to moderate retinitis that resulted in various degrees of destruction of the different layers, mononuclear cuffing of inner retinal vessels, proliferation of pigment epithelium, and choroiditis. Residually there was an absence of cellular elements in the atrophied areas of the retina, frequently a loss of layering and various numbers of pigment-containing cells. Moderately severe acute inflammation was seen in the retina of the fetus taken at 22 days after inoculation of its dam at 95 days. Ocular lesions did not occur in the other fetuses taken from heifers inoculated at earlier stages of gestation.     

Effects of averaging data series on the electroencephalographic response to noxious visceral stimulation in isoflurane-anaesthetized dogs

The effects of averaging epochs on electroencephalographic (EEG) responses to visceral stimulation has been determined in seven isoflurane-anaesthetized dogs. Quantitative EEG variables including 80% spectral edge frequency (SEF80), median frequency (MF), relative power in the delta, theta, alpha, beta band and power band ratios (theta/delta, alpha/delta, beta/delta) were recorded over 1min before stimulation and during a 1-minute stimulation period. During off-line analysis EEG variables were derived from either single 2-second EEG epochs or as an average from 5, 10, 15 and 30 consecutive 2-second epochs. Noxious stimulation resulted in significant increases in SEF80, MF, alpha power, beta power, alpha/delta ratio and beta/delta ratio. The number of variables that were significantly affected as well as the strength of changes as indicated by p-values, however, varied with the number of epochs subjected to averaging. The data suggest that stimulation-induced EEG changes may be more pronounced at lower rather than at higher averaging rates.     

Quantitative electroencephalographic findings in beagles anaesthetized with propofol

The purpose of this study was to assess quantitative electroencephalography (q-EEG) in 10 healthy beagle dogs under propofol anaesthesia in order to determine objective guidelines for diagnostic electroencephalographic (EEG) recordings and interpretation. The basic pattern after preliminary visual examination of EEG recordings was characterized by spindles, k-complexes, vertex sharp transients, and positive occipital transients that were superimposed on the slow background activity. The results of the q-EEG were characterized by the prevalence of slow rhythms delta and theta, both in absolute and relative power spectrum analysis, while fast rhythms (alpha and beta) were poorly represented. The distribution of single frequency bands was widespread for delta, focal for frontal and central for theta, as well as for most alpha and beta patterns. The present study has shown that the use of quantitative EEG gives information on the frequency content of the bio-electrical activity and defines the distribution of the single frequency bands under a standardized anaesthetic protocol.     

Protein kinase-C activity in phorbol myristate acetate-stimulated neutrophils from newborn and adult cattle.

Neutrophils from newborn calves have been shown to be deficient in ability to generate superoxide anion (O2-) after stimulation of the respiratory burst enzyme with the phorbol ester, phorbol 12-myristate 13-acetate (PMA). This compound activates the O(2-)-generating enzyme of bovine neutrophils through a pathway involving protein kinase C (PKC). To investigate the biochemical basis underlying this functional difference between neutrophils from newborn and adult cattle, we measured and compared the activity of the enzyme PKC in nonstimulated and PMA-stimulated bovine neutrophils. Neutrophils from newborn calves (n = 5) and adult cows (n = 5) were stimulated with various concentrations of PMA (0, 10, 100, and 500 ng/ml) for 3 minutes, and PKC activity was assayed in the cytosolic and the membrane fractions. In nonstimulated cells, most PKC activity was detected in the cytosolic fraction of neutrophils from newborn and adult cattle. Activity of PKC in the cytosol was dependent on the presence of added calcium and phospholipids, whereas membrane-associated PKC in nonstimulated cells did not have such dependence. Significant differences in PKC activity were not observed between newborn and adult cattle in either the cytosolic or the membrane fractions from nonstimulated cells. Stimulation with PMA caused redistribution of PKC activity in the cell (translocation) in newborns and adults, consisting of decrease in cytosolic PKC activity and increase in membrane-associated PKC activity. Similar to that in nonstimulated cells, PKC activity in cytosolic fractions from PMA-stimulated neutrophils was dependent on the presence of cofactors (calcium and phospholipids), whereas PKC activity in the membrane did not have such requirement.(ABSTRACT TRUNCATED AT 250 WORDS)     

Number and density of retinal photoreceptor cells with emphasis on oil droplet distribution in the Mallard Duck (Anas platyrhynchos var. domesticus)

This study was intended to determine the number and regional distribution of photoreceptor cells and different colored oil droplets in the retina of the Mallard Duck (Anas platyrhynchos var. domesticus). To estimate the number and density of photoreceptor cells, adult ducks were killed and both eyes were enucleated under deep anesthesia to prepare Nissl‐stained retinal whole‐mount samples. Different colored oil droplets were counted from color microphotographs of the freshly prepared retina. The mean number of retinal photoreceptors was approximately 6 308 828 ± 521 927, with a peak density of 33 573/mm² in the central retina. The density was similar in the nasal, temporal, ventral and dorsal areas of the retina. Five types of oil droplets were identified on the basis of color: red, orange, greenish‐yellow, yellow and clear. The mean density of oil droplets was highest in the central retina (17 639/mm²) and gradually declined towards the nasal, temporal, ventral and dorsal areas. The size of oil droplets gradually increased with retinal eccentricity and varied even within an area. The greenish‐yellow oil droplets were most abundant across the retina. Taken together, these results demonstrate the differential retinal distribution of photoreceptor cells and oil droplets in duck retina. We conclude that the area of high photoreceptor cell density, which is matched by high neuron densities of the ganglion cell layer, corresponds to the site of acute vision in duck retina.     

Protein kinase C (PKC) isotype profile in eosinophils from ponies with sweet itch and role in histamine-induced eosinophil activation

Eosinophils have been implicated in the pathogenesis of the seasonal equine allergic skin disease, sweet itch. Protein kinase C (PKC) is involved in regulating eosinophil function and antigen challenge has been reported to alter PKC isotype expression in blood eosinophils from allergic human subjects. Here we have compared the pattern of PKC isotype expression in eosinophils from sweet itch ponies with that in cells from normal ponies both during the active and inactive phases of the disease. A role for PKC in histamine-induced eosinophil activation was also investigated. Conventional PKCs alpha and beta, novel PKCs delta and epsilon and atypical PKCs iota and zeta were identified in eosinophils pooled from four allergic ponies during the inactive phase, when no clinical signs were evident. The PKC isotypes, like those in eosinophils from normal ponies, were located primarily in the particulate fraction of the cell. Isotype expression in cells from normal and allergic animals did not appear to be different. In contrast, during the active phase of the disease, when the sweet itch ponies had clinical signs, the expression of PKCs beta, epsilon and iota in eosinophils from these animals appeared to be increased relative to that in cells from normal ponies. When PKC expression in eosinophils from five individual normal and sweet itch ponies was compared, small, but statistically significant, increases in PKC epsilon and PKCdelta expression were evident in eosinophils from the sweet itch ponies during the active and inactive phases, respectively. The non-selective PKC inhibitors, staurosporine and Ro31-8220, significantly reduced histamine-induced superoxide production. Use of G?6976, an inhibitor of conventional PKCs, suggested that PKCalpha and/or beta were involved and that there was significantly greater inhibition of the response in eosinophils obtained from sweet itch ponies during the active phase. There was no significant difference in histamine-induced superoxide production by eosinophils from allergic and normal ponies and the functional significance of the increased PKC isotype expression in eosinophils from sweet itch ponies relative to that in cells from healthy animals remains to be established.     

Fine Structure of the Choriocapillaris, Bruch''s Membrane and Retinal Epithelium of the Cow

The fine structure of the choriocapillaris, Bruch's membrane and retinal epithelium was investigated in both the tapetal and non-tapetal fundus of the bovine eye. In ail locations the retinal epithelium consists of a single layer of cuboidal cells. The epithelial cells are joined laterally by apically located tight junctions and throughout the retina display numerous basal infoldings and fine apical processes which enclose rod outer segments. All retinal epithelial cells are rich in smooth endoplasmic reticulum and mitochondria and contain phagosomes. Although not as abundant, profiles of rough endoplasmic reticulum and polysomes are also noted in all locations. In non-tapetal areas, melano-somes are numerous whereas over the central tapetum fibrosum they are absent. The absence of melanosomes over a functional tapetum is to be expected. While lysosomes are present throughout the epithelial layer, over the tapetal region they appear to be more numerous. The apparent increase in lysosomal numbers in this location may indicate an enhanced shedding of outer segment material over the tapetum. Although some retinal epithelial cells are modified to accomodate a tapetum lucidum their morphology is basically similar throughout the retina and probably indicates that ail regions of the retinal epithelium are capable of the normal functions of this indispensible retinal layer. The choriocapillaris is heavily fenestrated on the border facing the retina and overlying the tapetum also displays fenestrae on its choroidal edge. Bruch's membrane ( complexus basalis ) is pentalaminate throughout the retina and is slightly thicker in the posterior fundus.     

N -ethyl- N -nitrosourea induces retinal photoreceptor damage in adult rats

Seven-week-old male Lewis rats received a single intraperitoneal injection of N-ethyl-N-nitrosourea (ENU) (100, 200, 400 or 600 mg/kg), and retinal damage was evaluated 7 days after the treatment. Sequential morphological features of the retina and retinal DNA damage, as determined by a TUNEL assay and phospho-histone H2A.X (γ-H2AX), were analyzed 3, 6, 12, 24 and 72 hr, 7 days, and/or 30 days after 400 mg/kg ENU treatment. Activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP) was analyzed immunohistochemically by poly (ADP-ribose) (PAR) expression in response to DNA damage of the retina. All rats that received ≥ 400 mg/kg of ENU developed retinal degeneration characterized by the loss of photoreceptor cells in both the central and peripheral retina within 7 days. In the 400 mg/kg ENU-treated rats, TUNEL-positive signals were only located in the photoreceptor cells and peaked 24 hr after ENU treatment. The γ-H2AX signals in inner retinal cells appeared at 24 hr and peaked at 72 hr after ENU treatment, and the PAR signals selectively located in the photoreceptor cell nuclei appeared at 12 hr and peaked at 24 hr after ENU treatment. However, degeneration was restricted to photoreceptor cells, and no degenerative changes in inner retinal cells were seen at any time points. Retinal thickness and the photoreceptor cell ratio in the central and peripheral retina were significantly decreased, and the retinal damage ratio was significantly increased 7 days after ENU treatment. In conclusion, ENU induced retinal degeneration in adult rats that was characterized by photoreceptor cell apoptosis through PARP activity.     

Functional and structural assessment of retinal sheet allograft transplantation in feline hereditary retinal degeneration

Purpose  To investigate whether sheets of fetal retinal allografts can integrate into the dystrophic Abyssinian cat retina with progressive rod cone degeneration.
Methods  Fetal retinal sheets (cat gestational day 42), incubated with BDNF microspheres, were transplanted to the subretinal space of four cats at an early disease stage. Cats were studied by fundus examinations, bilateral full-field flash ERGs, and indocyanine green and fluorescein angiograms up to 4 months following surgery. E42 donor and transplanted eyes were analyzed by histology and immunohistochemistry for retinal markers.
Results  Funduscopy and angiography showed good integration of the transplants in two of four cats, including extension of host blood vessels into the transplant and some scarring in the host. In these two, transplants were found in the subretinal space with laminated areas, with photoreceptor outer segments in normal contacts with the host retinal pigment epithelium. In some areas, transplants appeared to be well-integrated within the host neural retina. Neither of these two cats showed functional improvement in ERGs. In the other two cats, only remnants of donor tissue were left. Transplants stained for all investigated cellular markers. No PKC immunoreactivity was detected in the fetal donor retina at E42, but developed in the 4-month-old grafts.
Conclusions  Fetal sheet transplants can integrate well within a degenerating cat retina and develop good lamination of photoreceptors. Functional improvement was not demonstrated by ERG in cats with well-laminated grafts. Transplants need to be further evaluated in cat host retinas with a more advanced retinal degeneration using longer follow-up times.