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Analgesic effects of cranial laser treatment in two rat nociception models.

The present experiments sought to establish dose dependency and time course for effects of cranial laser irradiation in two rodent models of pain. These were the hot plate and tail flick tests, which are both widely used to quantify analgesic drug effects. The laser used was an Omega Biotherapy 3ML (wavelength 820 nM, average power output 100 mW, pulse frequency 5 kHz) and irradiation was applied to rats' shaved heads above the midbrain. In the first experiment, four groups of 10 rats received doses of 0, 6, 12, 18, and 24 J/cm2 in random orders prior to hot plate testing either immediately, 30 min, 1 h or 24 h postlaser. The second study employed three groups of 10 rats receiving 0, 12, and 18 J/cm2 in random orders prior to tail flick testing at the three shorter times above. Latency to lick hind paws on the hot plate was highly significantly prolonged by laser treatment across all doses and time periods, F(4, 126) = 4.51, p < 0.01. There was good dose dependency for immediate observations, but at 24 h 18 J/cm2 was the most effective dose. Laser treatment also delayed tail flick responses at both doses and all time periods, F(2, 54) = 10.60, p < 0.001, but 12 and 18 J/cm2 doses were similar in efficacy.

Analgesia↗

Skeletal muscle myosin is the autoantigen for experimental autoimmune myositis.

Experimental autoimmune myositis (EAM) is a rodent model for human inflammatory muscle disease (IMD). It can be induced by immunization of rodents with skeletal muscle homogenate and adjuvant. The specific myositogenic autoantigen has not been clearly identified although some evidence points to skeletal muscle myosin. In this report we strengthen this evidence, showing that Lewis rats immunized with purified skeletal muscle myosin develop EAM with the same pattern and severity as EAM induced by whole rabbit skeletal muscle homogenate (WRM). Multiple inflammatory lesions are detected histopathologically in the biceps, quadriceps, and gastrocnemius muscles. Myosin-reactive T cells from animals immunized either with myosin or with WRM have similar patterns of antigen-induced proliferation. The results show that myosin, a component of skeletal muscle, is at least one autoantigen in EAM.

Animals↗

Gene delivery to the eye using adeno-associated viral vectors.

Adeno-associated virus (AAV) vectors provide a useful way to deliver genes to the eye. They have a number of important properties which make them suitable for this purpose, not least their lack of significant pathogenicity and the potential for long-term transfection of retinal cells. The optimal methods for AAV-mediated gene delivery are determined by the location and characteristics of the target cell type. Efficient gene delivery to photoreceptors and pigment epithelial cells following subretinal injection of AAV has been achieved in various animal models. AAV-mediated gene therapy has been shown to slow photoreceptor loss in rodent models of primary photoreceptor diseases and in dogs with a naturally occurring disease similar to human Leber's congenital amaurosis (LCA). Efficient gene delivery to other cell types such as retinal ganglion cells (RGCs), however, has been more problematic. In this article, we review the potential uses of AAV-mediated gene delivery to the eye. We describe the selection of an appropriate AAV vector for ocular gene transfer studies and discuss the techniques used to deliver the virus to the eye and to assess ocular transfection. We emphasize our techniques for successful gene transfer to retinal ganglion cells, which have often proven challenging to transfect with high efficiency. Using a modified AAV incorporating a chicken beta-actin (CBA) promoter and the woodchuck hepatitis posttranscriptional regulatory element, we describe how our techniques allow approximately 85% of rat retinal ganglion cells to be transfected within 2 weeks of a single intravitreal virus injection. Our techniques facilitate the study of the pathogenesis of RGC diseases such as glaucoma and the development of novel new treatments based on gene therapy.

Adenoviridae↗

Modeling biological variability in 2-D gel proteomic carcinogenesis experiments.

We propose a statistical method to model the underlying distribution of protein spot volumes in 2-D gels using a generalized model (GM). We apply this approach to discover mechanisms of chemical carcinogenesis in a rodent model. We generated 247 protein spots that were common to all gels (n = 18). Traditional statistical methods found 6.5% (13 out of 247) significant protein spots, our GM approach yielded a total of 53 (22.5%) differentially expressed protein spots.

9,10-Dimethyl-1,2-benzanthracene↗

T helper 1 and T helper 2 cells are pathogenic in an antigen-specific model of colitis.

Dysregulated T cell responses to enteric bacteria have been implicated as a common mechanism underlying pathogenesis in rodent models of colitis. However, the bacterial species and T cell specificities that induce disease have been poorly defined. We have developed a model system in which target antigen, bacterial host, and corresponding T cell specificity are defined. OVA-specific T cells from DO11.RAG-2(-/-) TCR transgenic mice were transferred into RAG-2(-/-) recipients whose intestinal tracts were colonized with OVA-expressing or control Escherichia coli. Transfer of antigen-naive DO11.RAG-2(-/-) T cells into recipients colonized with OVA-E. coli resulted in enhanced intestinal recruitment and cell cycling of OVA-specific T cells; however, there was no development of disease. In contrast, transfer of polarized T helper (Th) 1 and Th2 populations resulted in severe wasting and colitis in recipients colonized with OVA-expressing but not control E. coli. The histopathologic features of disease induced by Th1 and Th2 transfers were distinct, but disease severity was comparable. Induction of disease by both Th1 and Th2 transfers was dependent on bacterially associated OVA. These results establish that a single bacterially associated antigen can drive the progression of colitis mediated by both Th1 and Th2 cells and provide a new model for understanding the immunoregulatory interactions between T cells responsive to gut floral antigens.

Adoptive Transfer↗

Rat model for human cryptosporidiosis.

Effective treatment for Cryptosporidium infection in immunocompromised patients has yet to be found. We report a rodent model of persistent Cryptosporidium infection. Sprague-Dawley rats were injected subcutaneously twice a week for 8 weeks with 25 mg of hydrocortisone acetate. Fed a regular low-protein diet for 9 weeks, they were challenged once with 10(5) calf Cryptosporidium oocysts 5 weeks after the start of the hydrocortisone acetate regimen. Oocyst shedding was evaluated in feces daily by using a carbolfuchsin-staining method. Rats shed oocysts from days 2 to 9 after ingestion and developed a persistent infection for more than 38 days. Excretion was lower after subsequent parasite challenges, suggesting that a degree of protection developed progressively. The results suggest that this experimental model provides a procedure for screening candidate therapeutic agents.

Animals↗

A new animal model for modulating myosin isoform expression by altered mechanical activity.

The purpose of this study was to develop a new rodent model that is capable of delineating the importance of mechanical loading on myosin heavy chain (MHC) isoform expression of the plantar and dorsi flexor muscles of the ankle. The essential components of this system include 1) stimulating electrodes that are chronically implanted into a muscle, allowing for the control of the activation pattern of the target muscle(s); 2) a training apparatus that translates the moment of the ankle into a linear force; and 3) a computer-controlled Cambridge 310 ergometer. The isovelocity profile of the ergometer ensured that the medial gastrocnemius (MG) produced forces that were > 90% of maximal isometric force (Po), and the eccentric contractions of the tibialis anterior (TA) were typically 120% of Po. Both the concentric and eccentric training programs produced statistically significant increases in the muscle mass of the MG (approximately 15%) and TA (approximately 7%) as well as a decrease in myofibrillar adenosinetriphosphatase activity. Both the white and red regions of the MG and TA exhibited significant increases in the relative content of the type IIa MHC and concomitant decreases in type IIb MHC expression. Although the red regions of the MG and red TA contained approximately 10% type I MHC, the training programs did not affect this isoform. It appears that when a fast-twitch muscle is stimulated at a high frequency (100 Hz) and required to contract either concentrically or eccentrically under high loading conditions, the expression of the type IIa MHC isoform will be upregulated, whereas that of the type IIb MHC will be concomitantly downregulated.

Adenosine Triphosphatases↗

Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function in ruminants.

The oocyte-secreted polypeptide growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, also known as GDF9B) have both been shown to be essential for ovarian follicular development and ovulation rate. In addition, it is known from both in vivo and in vitro studies that these factors co-operate in some manner. To date, most studies examining the in vitro effects of these growth factors have used the rodent model. However, the evidence suggests that these growth factors have somewhat different roles between rodents and ruminants. Therefore, the objectives of these studies were to examine the effects of GDF9 and BMP15, alone and together, on the functions of ovine and bovine granulosa cells under in vitro conditions. Ovine (o)BMP15 given together with murine (m)GDF9 or oGDF9 was more potent in stimulating (3)H-thymidine incorporation by ovine granulosa cells compared with each growth factor alone. For bovine granulosa cells, there appeared to be little or no co-operativity between oBMP15 and oGDF9 as oBMP15 alone was as potent as any combination of the two growth factors in stimulating (3)H-thymidine uptake. The species of origin of GDF9 affected the progesterone response in ovine granulosa cells with mGDF9 stimulating and oGDF9 inhibiting progesterone production. Ovine BMP15 alone had no effect on progesterone production by ovine granulosa cells and these growth factors did not appear to co-operate. FSH-stimulated progesterone production by bovine granulosa cells was most potently inhibited when oBMP15 and murine or ovine GDF9 were administered together. As was observed for progesterone, the species of origin of GDF9 affected inhibin production by ovine granulosa cells where mGDF9 inhibited while oGDF9 stimulated production. Murine GDF9 also inhibited inhibin production from bovine granulosa cells. For both ovine and bovine granulosa cells, BMP15 alone had no effect on inhibin production and there did not appear to be any co-operation between GDF9 and BMP15. These results indicate that the effects of BMP15 and GDF9 varied with respect to the species of origin of the growth factor. Moreover, the effects of GDF9 and BMP15 together were often co-operative and not always the same as those observed for these growth factors alone.

Animals↗

Mutations in the promoter of adenylyl cyclase (AC)-III gene, overexpression of AC-III mRNA, and enhanced cAMP generation in islets from the spontaneously diabetic GK rat model of type 2 diabetes.

Glucose-induced insulin release is decreased in the spontaneously diabetic GK rat, a nonobese rodent model of type 2 diabetes. Forskolin restores the impaired insulin release in both the isolated perfused pancreas and isolated islets from these rats (Abdel-Halim et al., Diabetes 45:934-940, 1996). We demonstrate here that the insulinotropic effect of forskolin in the GK rat is due to increased generation of cAMP and that it is associated with overexpression of adenylyl cyclase (AC)-III mRNA and gene mutations. The AC-III mRNA overexpression was demonstrated by in situ hybridization using oligonucleotide probes binding to different regions of the rat AC-III mRNA. It was associated with the presence of two point mutations identified at positions -28 bp (A --> G) and -358 bp (A --> C) of the promoter region of the AC-III gene and was demonstrable in both GK rat islets and peripheral blood cells. Transfection of COS cells with a luciferase reporter gene system revealed up to 25-fold increased promoter activity of GK AC-III promoter when compared with normal rat promoter (P < 0.0001). In conclusion, forskolin restores the impaired insulin release in islets of the GK rat through enhanced cAMP generation. This is linked to overexpression of AC-III mRNA in GK islets due to two functional point mutations in the promoter region of the AC-III gene.

Adenylyl Cyclases↗

Nonconvulsive electrocorticographic paroxysms (absence epilepsy) in rat strains.

Rat strains were screened for evidence of unresponsive periods associated with high-voltage spike and wave paroxysms on electroencephalography--a rodent model of human absence epilepsy. Five commonly used strains were newly noted to express the spike and wave phenomenon. Only an inbred Sprague Dawley rat strain did not exhibit such episodes. The existence of the phenomenon in many and unrelated inbred rat strains suggests that both genetic and environmental factors are causal.

Animals↗

Management, breeding, and reproductive performance of pine voles.

Pine voles (Microtus pinetorum) serve as an interesting rodent model for studies of fossorial adaptation and mammalian mating systems. They are unusual among rodents in displaying a cooperative system of breeding. Here we describe the management of a breeding colony of pine voles. This species is relatively easy to maintain in the laboratory, although care must be taken to provide sufficient water in their diet. Details of reproduction, as well as growth and development of the young, are included. Pine voles have a smaller litter size (2.3 +/- 0.9 pups at birth) than do other arvicoline rodents but are still prolific breeders, producing, on average, a litter per month for more than 12 months. These rodents are useful for comparison with other arvicoline rodents, as well as providing an opportunity to investigate the behavior, physiology, and reproduction of an easily managed cooperatively breeding mammal.

Aging↗

Plasmodium yoelii uses the murine Duffy antigen receptor for chemokines as a receptor for normocyte invasion and an alternative receptor for reticulocyte invasion.

Erythrocyte invasion by malaria parasites is a complex multistep process involving parasite and erythrocyte receptors. It is a critical stage in the parasite life cycle and, therefore, a logical step in which to intervene to prevent or ameliorate disease. Rodent models of malaria, commonly Plasmodium yoelii, are frequently used for studies of malaria pathogenesis. Little is known, however, about the invasion machinery of rodent malaria parasites. We have found previously that mice congenic for a region of chromosome 1, containing the Duffy antigen/receptor for chemokines (DARC), have different susceptibility to P yoelii infection. Because P vivax, a human parasite, and P knowlesi, a simian parasite, use DARC to enter human erythrocytes, we sought to identify the role of the murine DARC in P yoelii invasion. Using a novel in vivo invasion assay and DARC knock-out mice, we found that DARC knock-out normocytes (mature erythrocytes) had negligible levels of P yoelii invasion compared with wild-type normocytes, demonstrating that DARC is a receptor for invasion of murine erythrocytes. In contrast, DARC knock-out reticulocytes were invaded at a rate similar to that for wild-type reticulocytes. We conclude that there is a DARC- independent pathway for reticulocyte invasion. These findings represent the first identification of a murine malaria receptor on erythrocytes and the first determination that different pathways of invasion exist on normocytes and reticulocytes. Because we show conservation of host-receptor interactions between rodent and human malaria, we can now use this model to identify how immunity can interfere with the invasion process.

Animals↗

Microglial activation varies in different models of Creutzfeldt-Jakob disease.

Progressive changes in host mRNA expression can illuminate crucial pathogenetic pathways in infectious disease. We examined general and specific approaches to mRNA expression in three rodent models of Creutzfeldt-Jakob disease (CJD). Each of these models displays distinctive neuropathology. Although mRNAs for the chemokine receptor CCR5, the lysosomal protease cathepsin S, and the pleiotropic cytokine transforming growth factor beta1 (TGF-beta1) were progressively upregulated in rodent CJD, the temporal patterns and peak magnitudes of each of these transcripts varied substantially among models. Cathepsin S and TGF-beta1 were elevated more than 15-fold in mice and rats infected with two different CJD strains, but not in CJD-infected hamsters. In rats, an early activation of microglial transcripts preceded obvious deposits of prion protein (PrP) amyloid. However, in each of the three CJD models, the upregulation of CCR5, cathepsin S, and TGF-beta1 was variable with respect to the onset of PrP pathology. These results show glial cell involvement varies as a consequence of the agent strain and species infected. Although neurons are generally assumed to be the primary sites for agent replication and abnormal PrP formation, microglia may be targeted by some agent strains. In such instances, microglia can both process PrP to become amyloid and can enhance neuronal destruction. Because microglia can participate in agent clearance, they may also act as chronic reservoirs of infectivity. Finally, the results here strongly suggest that TGF-beta1 can be an essential signal for amyloid deposition.

Animals↗

The immune response and the evaluation of acquired immunity against gastrointestinal nematodes in cattle: a review.

The present review discusses the immune responses to gastrointestinal nematodes in cattle and the different immunological and parasitological parameters used to assess acquired immunity. Measuring acquired immunity to gastrointestinal nematodes in cattle (e.g. for the evaluation of candidate parasite vaccines) is hampered by the limited understanding of bovine immune responses against gastrointestinal parasites. In this paper the available data on protective immunity against gastrointestinal nematodes, and especially Ostertagia ostertagi, in cattle are compared with the current knowledge of protective immune responses against gastrointestinal nematodes in rodent models and small ruminants. In contrast to the immune response in mice, which is controlled by T helper 2 (Th2) lymphocytes and results in mast cell- or goblet cell-mediated expulsion of adult worms, bovine immune responses to O. ostertagi do not show a clear Th2 cytokine profile, nor do they result in rapid expulsion of the parasite. The first manifestation of immunity to O. ostertagi in calves is a reduction of worm fecundity, possibly regulated by the local IgA response. Worm numbers are only reduced after a prolonged period of host-parasite contact, and there are indications that O. ostertagi actively suppresses the host's immune response. Until the mechanisms of protective immunity against O. ostertagi are revealed, the use of immunological parameters to estimate acquired immunity in cattle is based on their correlation with parasitological parameters and on extrapolation from rodent and small ruminant models. Assessing the resistance of calves against a challenge infection by means of parasitological parameters is probably still the most accurate way to measure acquired immunity against gastrointestinal nematodes.

Animals↗

Acute dystonia induced by neuroleptic drugs.

About 2.5% of patients treated with neuroleptic drugs develop acute dystonia within 48 h of commencing therapy. The symptoms remit on drug withdrawal or following anticholinergic therapy. Acute dystonia can also be reliably induced in many primate species by neuroleptic treatment with comparable time course, symptomatology and pharmacological characteristics to those observed in man. In general, New World monkeys appear more susceptible to acute dystonia than Old World primates. It is at present not clear whether all primates, including man, would exhibit dystonia if a sufficiently high dose of neuroleptic was administered. Alternatively, some unknown, possibly species-specific or even genetic, factors may determine an individual's susceptibility to develop dystonia. Use of a rodent model of dystonia might enable more detailed analysis of biochemical correlates of dystonic behaviour. Whilst rodents do not exhibit overt dystonic behaviour after neuroleptic treatment, they may develop oral dyskinesias which bear a close pharmacological similarity to dystonia in man and primates. However, it is not known whether chewing induced by neuroleptic drugs in rats resembles acute dystonia in primates or whether this is another movement disorder possibly unique to rodent species. The pathophysiology of acute dystonia remains unknown, but may involve striatal dopaminergic and cholinergic function. In view of the close similarity between dystonia in man and other primates, studies on the mechanisms whereby neuroleptic drugs cause acute dystonic reactions in monkeys may give some clues to the pathogenesis of spontaneous dystonia in man.

Acute Disease↗

PGE2 receptors rescue motor neurons in a model of amyotrophic lateral sclerosis.

Recent studies suggest that the inducible isoform of cyclooxygenase, COX-2, promotes motor neuron loss in rodent models of ALS. We investigated the effects of PGE2, a principal downstream prostaglandin product of COX-2 activity, on motor neuron survival in an organotypic culture model of ALS. We find that PGE2 paradoxically protects motor neurons at physiological concentrations in this model. PGE2 exerts its downstream effects by signaling through a class of four distinct G-protein-coupled E-prostanoid receptors (EP1-EP4) that have divergent effects on cAMP. EP2 and EP3 are dominantly expressed in ventral spinal cord in neurons and astrocytes, and activation of these receptor subtypes individually or in combination also rescued motor neurons. The EP2 receptor is positively coupled to cAMP, and its neuroprotection was mimicked by application of forskolin and blocked by inhibition of PKA, suggesting that its protective effect is mediated by downstream effects of cAMP. Conversely, the EP3 receptor is negatively coupled to cAMP, and its neuroprotective effect was blocked by pertussis toxin, suggesting that its protective effect is dependent on Gi-coupled heterotrimeric signaling. Taken together, these data demonstrate an unexpected neuroprotective effect mediated by PGE2, in which activation of its EP2 and EP3 receptors protected motor neurons from chronic glutamate toxicity.

Alprostadil↗

Toxicity and dosimetry of (177)Lu-DOTA-Y3-octreotate in a rat model.

Radiolabeled somatostatin analogs have demonstrated effectiveness for targeted radiotherapy of somatostatin receptor-positive tumors in both tumor-bearing rodent models and humans. A radionuclide of interest for cancer therapy is reactor-produced (177)Lu (t(1/2) = 6.64 d; beta(-) [100%]). The high therapeutic efficacy of the somatostatin analog (177)Lu-DOTA-Tyr(3)-octreotate (DOTA-Y3-TATE, where DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) was previously demonstrated in a tumor-bearing rat model (Erion et al., J. Nucl. Med. 1999;40:223P; de Jong et al., Int. J. Cancer, 2001; 92:628-633). In the current study, the toxicity and dosimetry of (177)Lu-DOTA-Y3-TATE were determined in both normal and tumor-bearing rats. Doses of (177)Lu-DOTA-Y3-TATE ranging from 0 to 123 mCi/kg were administered to rats and complete blood counts (CBCs) and blood chemistries were analyzed out to 6 weeks. No overt signs of toxicity were observed with (177)Lu-DOTA-Y3-TATE (i.e., lethargy, weight loss, scruffy coat or diarrhea) at any of the dose levels. Blood chemistries and CBCs were normal except for the white blood cell counts, which showed a dose-dependent decrease. The maximum tolerated dose was not reached at 123 mCi/kg. The biodistribution of (177)Lu-DOTA-Y3-TATE was determined in CA20948 rat pancreatic tumor-bearing rats, and the data were used to estimate human absorbed doses to normal tissues. The dose-limiting organ was determined to be the pancreas, followed by the adrenal glands. The absorbed dose to the rat CA20948 tumor was estimated to be 336 rad/mCi (91 mGy/MBq). These data demonstrate that (177)Lu-DOTA-Y3-TATE is an effective targeted radiotherapy agent at levels that show minimal toxicity in this rat model.

Animals↗

Different types of segmental sclerosing glomerular lesions in six experimental models of proteinuria.

From 133 to 615 glomeruli were examined in sections of kidneys from each of 60 animals, representing six rodent models of proteinuria. Particular attention was paid to the position of segmental lesions. Lewis rats given sheep anti-rat glomerular basement membrane antibodies had lesions almost exclusively at the glomerulo-tubular junction. Wistar rats on a diet of 24 per cent casein or with subtotal nephrectomy and a diet of 24 per cent soya had lesions mainly at the hilum. Wistar rats given bovine serum albumin had global lesions but virtually no segmental lesions. Wistar rats given puromycin aminonucleoside had lesions at the glomerulo-tubular junction and global mesangial abnormalities shortly after the treatment but later developed segmental lesions at all parts of the glomerulus. Untreated BUF/Mna rats had lesions at the glomerulo-tubular junction early in life but later had lesions at all parts of the glomerulus. Untreated NZB/NZW hybrid mice had various types of glomerulonephritis and also had lesions at the glomerulo-tubular junction. These findings showed that (1) segmental lesions at the glomerulo-tubular junction, or glomerular tip, occur in experimental animals, a fact not previously reported, and these tip changes are a common feature in several different models of proteinuria; (2) hilar segmental lesions are seen in conditions with hyperfiltration of protein; and (3) segmental lesions at various parts of the glomerulus are seen in some models of proteinuria and probably indicate late effects of random toxic damage to the glomerulus. Thus, there are at least three different types of segmental glomerular lesions in experimental animals--tip, hilar, and random--with different morphology and pathogenesis. It is likely that these findings can be extended to human renal diseases with segmental glomerular lesions. This will help to clarify the controversial and unsatisfactory term focal segmental glomerulosclerosis.

Animals↗