Repeated microsphere injections in rats

There is currently some question concerning the dose of microspheres and blood sample withdrawal rates which will give accurate reproducable tissue blood flow measurements. In these experiments unanesthetized male Sprague-Dawley rats were tested with repeated injections of 100,000 15±5μ microspheres to monitor the effect on cardiovascular and regional hemodynamic measurements. No significant change in blood pressure, cardiac output or tissue blood flow was seen with up to 3 repeated injections of 100,000 microspheres per injection. In addition, no difference was observed between blood sample withdrawal rates of 0.4 or 0.8 ml/min. These data are consistent with previous reports that over 300,000 microspheres can be injected into the rat with no measurable change in hemodynamics and that accurate tissue blood flow measurements are dependent on an adequate number of microspheres being trapped in the reference blood and tissue samples rather than the rate of blood withdrawal.     

Two novel connexin32 mutations cause early onset X-linked Charcot-Marie-Tooth disease

Background  

X-linked Charcot-Marie Tooth (CMT) is caused by mutations in the connexin32 gene that encodes a polypeptide which is arranged in hexameric array and form gap junctions.     

Cardiac abnormalities cause early lethality of jumonji mutant mice

jumonji (jmj) mutant mice, obtained by a gene trap strategy, showed several morphological abnormalities including neural tube and cardiac defects, and died in utero around embryonic day 11.5 (E11.5). It is unknown what causes the embryonic lethality. Here, we demonstrate that exogenous expression of jmj gene in the heart of jmj mutant mice rescued the morphological phenotypes in the heart, and these embryos survived until E13.5. These results suggest that there are at least two lethal periods in jmj mutant mice, and that cardiac abnormalities may cause the earlier lethality. In addition, the rescue of the cardiac abnormalities by the jmj transgene provided solid evidence that the cardiac abnormalities resulted from mutation of the jmj gene.     

Retro-orbital injections in mice

Intravenous vascular access is technically challenging in the adult mouse and even more challenging in neonatal mice. The authors describe the technique of retro-orbital injection of the venous sinus in the adult and neonatal mouse. This technique is a useful alternative to tail vein injection for the administration of non-tumorigenic compounds. The authors report that they have routinely used this technique in the adult mouse to administer volumes up to 150?μl without incident. Administration of retro-orbital injections is more challenging in neonatal mice but can reliably deliver volumes up to 10?μl.     

Repeated injections of PEG-PE liposomes generate anti-PEG antibodies

Liposomes containing the polyethylene glycol (PEG) derivative of phosphatidyl ethanolamine (PE) have recently been found to be promising drug carriers, as they facilitate controlled and target-oriented release of therapeutics. They also reduce the side effects of many drugs. Here, we present the results of a study on antiliposomal properties of rabbit sera obtained after weekly injections of small liposomes containing 20% PEG-PE. The effect was analysed as the level of induced carboxyfluorescein release from these liposomes in vitro. The incubation of liposomes with rabbit serum taken after the injections induced the release of carboxyfluorescein at a higher level than was seen for incubation with untreated animal's serum. The strongest effect was observed for serum obtained after the second injection, i.e. during the second week of the study. The effect was much smaller after the serum samples were preheated at 56 degrees C. The binding of serum proteins by PEGylated liposomes was analysed via gel filtration and via the immunoblot technique using goat anti-rabbit IgG; this revealed that the serum protein which bound to the liposomes in vitro had a molecular weight of 55 kD and reacted with the anti-IgG antibody. Competition with PEG or lipids indicate that this IgG has an anti-PEG activity. We therefore assume that these antibodies are responsible for the activation of complement and leakage induction of PEG-liposomes. Such antibodies could be responsible for increased phagocytosis by RES macrophages (in particular liver macrophages) and decreased circulation time.     

Regulated over-expression of DNA polymerase beta mediates early onset cataract in mice

Base excision repair (BER) is a tightly coordinated mechanism for repair of DNA base damage (via alkylation and oxidation) and base loss. From E. coli to yeast to human cells, subtle alterations in expression of BER proteins lead to mutagenic or genome instability phenotypes. DNA polymerase beta (beta-pol), the major BER polymerase, has been found to be over-expressed in human tumor tissues and more recently it has been shown that over-expression of beta-pol results in a mutator and genome instability phenotype. These previous reports imply that beta-pol over-expression is deleterious and suggests that such an imbalance may cause an overall functional deficiency in the BER pathway. In the present study, we have developed a bicistronic tetracycline-responsive transgenic system to over-express beta-pol in mice. We find that over-expression of beta-pol in the lens epithelium results in the early onset of severe cortical cataract, with cataractogenesis beginning within 4 days after birth. In utero and post-natal suppression of transgenic Flag-beta-pol expression by doxycycline administration completely prevents cataract formation through adulthood, yet cataract is subsequently observed following removal of doxycycline and re-expression of the transgene. Cataract development accompanies increased expression of cyclooxygenase-2 in the lenticular fibers of the lens, implicating oxidative stress in the development of this cataractous phenotype. Although the mechanism for the transgene mediated cataractogenesis is not clear at this time, it is nevertheless intriguing that increased expression of beta-pol leads to such a phenotype. These results suggest that either a beta-pol expression imbalance negatively affects overall fidelity and/or BER capacity or that beta-pol has a role in lens epithelial cell differentiation.     

Advanced glomerulosclerosis is reversible in nephrotic mice

Advanced glomerulosclerosis, a common hallmark of chronic renal diseases (CRD) is believed to be irreversible, and it is thought that glomerular hyperfiltration and hypertrophy may participate in its pathogenesis. We demonstrate here that glomerulosclerosis is "reversible" in an animal model. We used nephrotic ICGN (nep/nep) mice which showed a rapid progression of glomerulosclerosis, accompanied by histological findings for glomerular hyperfiltration. It is known that ureter ligation reduces glomerular filtration in ligated kidneys. When ureter ligation was applied to our model, glomerulosclerosis (characterized by myofibroblast hyperplasia and over-accumulated matrix protein) weakened in conjunction with suppressed glomerular hypertrophy. During this process, glomerular myofibroblasts showed apoptotic cell death after unilateral ureter ligation (UUO) treatment. Our results suggest that inhibition of glomerular filtration in sclerotic tufts may cause glomerular remodeling through the modulation of molecular and cellular sclerogenesis.     

Evidence for early onset, polyclonal activation of T cell subsets in mice homozygous for lpr.

Mice homozygous for lpr and gld develop profound lymphadenopathy characterized by the accumulation of two functionally anergic T cell subsets, a predominant B220+CD4-CD8- double negative (DN) population and a minor, closely related CD4 dull+ B220+ population. Lymph nodes from diseased lpr and gld mice also contain abnormally high numbers of conventional T cells, and we reported recently that a high proportion of lpr and gld CD4+B220- T cells have the hallmarks of primed or memory T cells. In the present study, we further investigated the extent, ontogeny, and possible causes of T cell activation in lpr and gld mice. The criteria used to identify primed or memory T cells included activation-dependent increases in the expression of CD44, LFA-1, and the early activation Ag, CD69, and decreases in the expression of Mel-14 and CD45RB, as well as quantitative differences in the in vitro production of IFN-gamma and the TNF-alpha by stimulated cells. A comparison of TCR V beta gene utilization by lpr T cell subsets also was undertaken. The results showed that T cell activation was widespread and complex. CD8+ T cells exhibited a similar pattern of activation to CD4+B220- T cells. The activation of these two subsets occurred in parallel, was in evidence by 4 to 6 wk of age, and was both chronic and progressive. The proportions of CD44hiLFA-1hi, CD4+B220-, and CD8+ T cells increased steadily between 4 and 20 wk of age, but changes in T cell growth, Mel-14, and CD45RB expression and cytokine secretion were not observed until mice were older than 11 wk. A very different pattern of activation was observed for B220+ T cells. At all ages, B220+ DN and CD4+B220+ T cells were CD44hiMel-14hi and 60 to 75% were CD69+. The expression of CD69 appeared to be stimulus dependent rather than constitutive, suggesting that these cells, too, may be chronically stimulated in vivo. In keeping with their anergic state, DN T cells responded poorly to cross-linking of CD69. The stimuli inducing chronic activation of CD4+B220- and CD8+ T cells are unlikely to include inappropriate reactions to autoantigens because there was no evidence for selective accumulation of CD4+ or CD8+ T cells bearing particular V beta genes or potentially self-reactive cells that normally are deleted in the thymus. By comparison, C3H-lpr DN cells displayed some potentially significant differences in V beta 6 and V beta 9 expression from CD4+B220- and CD8+ T cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Repeated daily injections and osmotic pump infusion of isoproterenol cause similar increases in cardiac mass but have different effects on blood pressure

We found in mice that repeated single daily subcutaneous (s.c.) isoproterenol (ISO) injections, like constant infusions using osmotic minipumps, caused increased biventricular mass or weight relative to body weight (VW/BW). We found that 5 (1/d) s.c. injections of 2, 10, or 20 microg/g body weight caused equivalent VW/BW increases as compared with 5-d infusions at 20 microg/(g.d)). While it is often presumed that ISO elicits hypertrophy by a direct effect on the myocytes, growth may also be secondary to systemic hemodynamic effects. The 2 modes of ISO administration had different effects on mean arterial blood pressure (MABP) and heart rate. Using telemetry we observed that single injections of ISO (0, 0.5, 2, and 10 microg/g) were associated with hypotension and tachycardia with a duration but not a magnitude that was dose dependent. MABP dropped rapidly to 60 mm Hg for more than 2 h at the highest dose. Constant s.c. infusion of ISO at 20 microg/(g.d) initially lowered MABP to about 70 mm Hg for 24 h. At 48 h MABP was normal, but rose 10 mm Hg higher than baseline by day 5. Thus, different routes of administration of ISO that cause comparable increases in VW/BW had different effects on MABP. Thus when evaluating mouse models of ISO-induced cardiac hypertrophy, both repeated daily injections or infusions can cause similar increases in VW/BW, but the daily doses that are required are not the same. Furthermore, these different routes of administration have different hemodynamic sequelae and could potentially evoke different cardiac phenotypes.     

Doxorubicin-induced glomerulosclerosis with proteinuria in GFP-GABARAP transgenic mice

Autophagy is a process of cellular degradation, and its dysfunction elicits many pathological symptoms. However, the contribution of autophagy to kidney glomerular function has not been fully clarified. We previously reported that LC3, a promising executor of autophagy, played an important role in recovery from podocyte damage in an experimental nephrosis model (Asanuma K, Tanida I, Shirato I, Ueno T, Takahara H, Nishitani T, Kominami E, Tomino Y. FASEB J 17: 1165-1167, 2003). γ-Aminobutyric acid A receptor-associated protein (GABARAP), has recently been characterized as another homolog of LC3, although its precise role in autophagy remains unclear. We recently generated green fluorescent protein (GFP)-GABARAP transgenic mice, in which GFP-GABARAP is abundantly expressed in glomerular podocytes. We found that the transgenic mice showed no obvious phenotype, and podocytes isolated from these mice manifested autophagic activity almost equivalent to that of wild-type mice when measured in vitro. Surprisingly, a single injection of doxorubicin caused a greater increase in proteinuria and sclerotic glomeruli in transgenic mice compared with wild-type mice. Under these conditions, neither GFP-GABARAP nor endogenous GABARAP appeared to be recruited to autophagosomes, and both remained in the cytosol. Moreover, the cytosolic GFP-GABARAP was significantly colocalized with p62 to form aggregates. These results indicate that the GFP-GABARAP/p62 complex is responsible for impairment of glomerular function and that it retards recovery from the effects of doxorubicin.     

Circulating urokinase receptor as a cause of focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is a cause of proteinuric kidney disease, compromising both native and transplanted kidneys. Treatment is limited because of a complex pathogenesis, including unknown serum factors. Here we report that serum soluble urokinase receptor (suPAR) is elevated in two-thirds of subjects with primary FSGS, but not in people with other glomerular diseases. We further find that a higher concentration of suPAR before transplantation underlies an increased risk for recurrence of FSGS after transplantation. Using three mouse models, we explore the effects of suPAR on kidney function and morphology. We show that circulating suPAR activates podocyte β(3) integrin in both native and grafted kidneys, causing foot process effacement, proteinuria and FSGS-like glomerulopathy. Our findings suggest that the renal disease only develops when suPAR sufficiently activates podocyte β(3) integrin. Thus, the disease can be abrogated by lowering serum suPAR concentrations through plasmapheresis, or by interfering with the suPAR-β(3) integrin interaction through antibodies and small molecules targeting either uPAR or β(3) integrin. Our study identifies serum suPAR as a circulating factor that may cause FSGS.     

Dietary restriction prevents diabetogenic effect of streptozotocin in mice

The beneficial role of dietary restriction (DR) was studied in streptozotocin (STZ)-induced diabetes in mice. The DR mice exhibited the lower blood glucose (mg/dl) level as compared to ad libitum (AL) fed ones. After 3 months' DR, STZ treatment to both AL and DR mice showed significant (p < 0.001) elevation of the blood glucose level in AL-fed mice, while a lower level of glucose was maintained in DR-fed mice. The ability of maintaining a low blood glucose level in STZ-treated DR mice indicated that STZ might have been ineffective from its action on beta cells of pancreas by long-term DR. Thus, these findings suggested that DR may be an important tool for preventing the diabetic conditions. However, further studies are required to know the mechanism(s) of DR protection against diabetogenic action of STZ in experimental animals.     

Early induction of diabetes in NOD mice by streptozotocin

To clarify whether the non-obese diabetes prone (NOD) mouse has an unusual pancreatic sensitivity to damage, mice were administered streptozotocin in high dose (direct beta cell toxic) or multiple low-dose (autoimmune-insulitis generating) regimen. NOD mice were found to be less sensitive to the diabetogenic effects of high-dose streptozotocin than C57BL/6 mice, but were exquisitely responsive to the multiple low dose regimen when compared to C57BL/6 or C3H/HeJ mice. These results suggest that the basic defect in NOD mice resides in the immune system and that the NOD mouse may be a useful model to investigate the relationships between environmental factors and intrinsic genetic predisposition to diabetes.     

Intraneuronal Abeta causes the onset of early Alzheimer's disease-related cognitive deficits in transgenic mice

Progressive memory loss and cognitive dysfunction are the hallmark clinical features of Alzheimer's disease (AD). Identifying the molecular triggers for the onset of AD-related cognitive decline presently requires the use of suitable animal models, such as the 3xTg-AD mice, which develop both amyloid and tangle pathology. Here, we characterize the onset of learning and memory deficits in this model. We report that 2-month-old, prepathologic mice are cognitively unimpaired. The earliest cognitive impairment manifests at 4 months as a deficit in long-term retention and correlates with the accumulation of intraneuronal Abeta in the hippocampus and amygdala. Plaque or tangle pathology is not apparent at this age, suggesting that they contribute to cognitive dysfunction at later time points. Clearance of the intraneuronal Abeta pathology by immunotherapy rescues the early cognitive deficits on a hippocampal-dependent task. Reemergence of the Abeta pathology again leads to cognitive deficits. This study strongly implicates intraneuronal Abeta in the onset of cognitive dysfunction.     

Naloxone-precipitated jumping in mice pretreated with acute injections of opioids.

Naloxone induced jumping was examined in mice pretreated with single dose of narcotic agonist (morphine, heroin, LAAM, methadone), mixed agonist-antagonist (pentazocine, cyclazocine, buprenorphine), or an enkephalin analog (D-met², pro⁵)-enkephalinamide. Acute sensitization to naloxone, as demonstrated by jumping, was observed after pretreatment with the narcotics, the enkephalin analog, and to a lesser degree after cyclazocine and pentazocine. Mice pretreated with buprenorphine did not jump in response to naloxone. This procedure may be of value in the rapid identification of drugs with a propensity to produce morphine-like physical dependence.