Fasciola hepatica in the rat:immune responses associated with the development of resistance to infection

F. hepatica infections were established in rats and immune responses were monitored during primary and challenge infections. Antibody levels peaked at 3 weeks post-primary infection and at 6 days post-challenge infection. No significant correlation was found between antibody titre and number of flukes recovered at autopsy. Immunoblotting revealed a limited number of immunogenic polypeptides. When antibodies from these reactive bands were eluted and tested by IFA they all gave identical binding patterns: on juvenile fluke sections tegumental syncytium, tegumental cells and gut cells were labelled, while on adult sections the same antibodies labelled gut cells, reproductive tissue, excretory ducts and flame cells. This suggested that these tissues shared a common epitope or range of epitopes. A pronounced eosinophilia was observed throughout the infection period studied and infected liver sections showed massive cellular infiltration. Histochemical and immunocytochemical investigation of infected liver revealed the presence of large numbers of eosinophils, neutrophils, lymphocytes and phagocytes. The implications of these findings, to an understanding of concomitant immunity in the rat are discussed.

Rat clonidine mydriasis model:Imidazoline receptors are not involved

The clonidine mydriasis model in rats has been widely applied in preclinical research to characterize a -adrenoceptor antagonistic properties of drugs. The present study was undertaken to pharmacologically determine if imidazoline I receptors are also involved in this model system. Sigmoid dose-response curves for pupillary dilation were produced in pentobarbital anesthetized rats by intravenous administration of increasing doses of agonists (guanabenz for a -adrenoceptors, clonidine for both a - adrenoceptors and imidazoline I receptors, and rilmenidine for imidazoline I receptors). Two antagonists (RS 79948 for a -adrenoceptors and efaroxan for imidazoline I receptors) were used to antagonize the mydriasis elicited by those three agonists, with antagonistic potencies calculated. In additional experiments, we examined the effect of the selective imidazoline I receptor antagonist, AGN 192403, on clonidine-induced mydriasis. The results showed that pupillary response curves elicited by guanabenz, clonidine and rilmenidine were competitively antagonized by both RS 79948 (0.03-1 mg/kg) and efaroxan (0.03-1 mg/kg) in a dose-related fashion. The potencies of either antagonist against the three agonists were not significantly different. AGN 192403 (5 mg/kg) did not significantly shift the clonidine mydriasis curve. These results suggest that imidazoline I receptors are not functionally involved in the rat clonidine mydriasis model and support this in vivo system as a useful model for studies of a -adrenoceptors. © 2004 Elsevier B.V. All rights reserved.

Susceptibility of streptozotocin-induced diabetic rat retinal function and ocular blood flow to acute intraocular pressure challenge.

PURPOSE: To consider whether STZ-induced hyperglycemia renders rat retinal function and ocular blood flow more susceptible to acute intraocular pressure (IOP) challenge.

METHODS: Retinal function (electroretinogram, ERG) was measured during acute IOP challenge (10-100 mmHg, 5 mmHg increments, 3 min/step, vitreal cannulation) in adult Long-Evans rats (6-week old, citrate: n=6, STZ: n=10) 4 weeks after citrate buffer or streptozotocin (STZ, 65 mg/kg, blood glucose > 15 mmol/l) injection. At each IOP, dim and bright flash (-4.56, -1.72 log cd.s.m^-2) ERG responses were recorded to measure inner retinal and ON-bipolar cell function, respectively. Ocular blood flow (laser Doppler flowmetry, citrate; n=6, STZ; n=10) was also measured during acute IOP challenge. Retinae were isolated for qPCR analysis of nitric oxide synthase mRNA expression endothelial, eNos; inducible, iNos; neuronal, nNos).

RESULTS: STZ-induced diabetes increased the susceptibility of inner retinal (IOP at 50% response, 60.1, CI: 57.0-62.0 mmHg vs. citrate: 67.5, CI: 62.1-72.4 mmHg) and ON-bipolar cell function (STZ: 60.3, CI: 58.0-62.8 mmHg vs. citrate: 65.1, CI: 58.0-62.78 mmHg) and ocular blood flow (43.9, CI: 40.8-46.8 vs. citrate: 53.4, CI: 50.7-56.1 mmHg) to IOP challenge. Citrate eyes showed elevated eNos mRNA (+49.7%) after IOP stress, an effect not found in STZ-diabetic eyes (-5.7%, P<0.03). No difference was observed for iNos or nNos (P>0.05) following IOP elevation.

CONCLUSIONS: STZ-induced diabetes increased functional susceptibility during acute IOP challenge. This functional vulnerability is associated with a reduced capacity for diabetic eyes to upregulate eNOS expression and to autoregulate blood flow in response to stress.

Using the electroretinogram to understand how intraocular pressure elevation affects the rat retina

Intraocular pressure (IOP) elevation is a key risk factor for glaucoma. Our understanding of the effect that IOP elevation has on the eye has been greatly enhanced by the application of the electroretinogram (ERG). In this paper, we describe how the ERG in the rodent eye is affected by changes in IOP magnitude, duration, and number of spikes. We consider how the variables of blood pressure and age can modify the effect of IOP elevation on the ERG. Finally, we contrast the effects that acute and chronic IOP elevation can have on the rodent ERG.

Cytosolic phospholipase A2 gene expression in rat mesangial cells is regulated post-transcriptionally

Cytosolic phospholipase A2 (cPLA2) is thought to be the rate-limiting enzyme in the arachidonic acid/eicosanoid cascade. The ability of various agonists to increase steady-state cPLA2 mRNA levels has previously been reported. The current study delineates the contributions of transcriptional and post-transcriptional processes to the regulation of cPLA2 gene expression in response to a variety of agonists in cultured rat glomerular mesangial cells. Epidermal growth factor, platelet-derived growth factor, serum and phorbol myristate acetate all increase the half-life of cPLA2 mRNA transcripts, indicating a role for post-transcriptional modulation of gene expression. The presence of three ATTTA motifs in the 3' untranslated region (3'UTR) of the rat cPLA2 cDNA is ascertained. Heterologous expression of chimeric constructs with different 3'UTRs ligated into the 3' end of the luciferase coding region reveals that the presence of the cPLA2 3'UTR results in reduced luciferase activity compared with constructs without the cPLA2 3'UTR. Furthermore, the luciferase activity in the constructs with the cPLA2 3'UTR is increased in response to the same agonists which stabilize endogenous cPLA2 mRNA. A negligible effect of these agonists on transcriptional control of cPLA2 is evident using promoter-reporter constructs expressed in transient and stable transfectants. Taken together, these results indicate predominant post-transcriptional regulation of cPLA2 mRNA levels.

GABAergic control of arteriolar diameter in the rat retina

Purpose: To investigate the role of γ-aminobutryic acid (GABA) in the regulation of arteriolar diameter in the rat retina.

Methods.: The actions of GABA on arteriolar diameter were examined using ex vivo retinal whole-mount preparations and isolated vessel segments. In most experiments, arterioles were partially preconstricted with endothelin (Et)-1. The expression levels of GABAA and GABAB receptors on isolated rat retinal Müller cells were assessed by immunohistochemistry.

Results.: GABA (0.1–1 mM) evoked vasodilation or vasoconstriction of arterioles in whole-mount preparations. No such effects were observed with isolated vessel segments. In whole mount samples, the GABAA receptor agonist muscimol caused vasomotor responses in only a small proportion of vessels. In contrast, arteriolar responses to the GABAB receptor agonists baclofen and SKF97541 more closely resembled those observed with GABA. No responses were seen with the GABAC receptor agonist 5-methylimidazoleacetic acid. GABA-induced vasodilator responses were, for the most part, repeatable in the presence of the GABAA receptor antagonist bicuculline. These responses, however, were completely blocked in the presence of the GABAB receptor inhibitor 2-hydroxysaclofen. Strong immunolabeling for both GABAA and GABAB receptors was detected in isolated Müller cells. In the absence of Et-1–induced preconstriction, most vessels were unresponsive to bicuculline or 2-hydroxysaclofen.

Conclusions.: GABA exerts complex effects on arteriolar diameter in the rat retina. These actions appear largely dependent upon the activation of GABAB receptors in the retinal neuropile, possibly those located on perivascular Müller cells. Despite these findings, endogenous GABA appears to contribute little to the regulation of basal arteriolar diameter in the rat retina.

Effects of thyroxine treatment on histology and behavior using the methimazole model of congenital hypothyroidism in the rat

The timing of thyroxine (T4) replacement treatment in congenital hypothyroidism (CH) has been suggested to be important for optimizing cognitive recovery in humans; however this has not been fully established using modern animal models of CH. Consequently, the current studies investigated the ameliorating effects of postnatal T4 treatment on neuropathology and behavior in CH rats. Rat dams were administered methimazole to produce CH offspring, then brain tissue from male CH pups was analyzed to determine the effects of postnatal (P3, P7, P14 and P21) T4 treatment on hippocampal dendritic branching and the expression of nerve growth factor (NGF). Two operant behavioral procedures were employed to confirm and extend previous findings obtained using this model, and to investigate timelines for instigating T4 treatment on improved behavioral outcomes. T4 treatment initiated at P14 was protective of a reduction in dendritic branching in the hippocampus, and initiated at P7 was protective of a reduction of NGF expression in the fimbria of the hippocampus. Induction of CH did not affect the acquisition of simple operant response rules but had a significant effect on the acquisition of complex operant rules subsequently imposed. Furthermore, T4 treatment initiated at P3 protected learning deficits seen following the imposition of complex operant response rules. These findings indicate T4 treatment initiated at P7 is sufficient for the protection of hippocampal NGF expression and dendritic branching but for the protection of complex behavioral abilities T4 treatment is necessary prior to or approximating P3.