Refinement of intrathymic injection in mice

Direct intrathymic injection is a common procedure used in several types of experimental protocols in the mouse. Currently available approaches involve major surgical procedures that expose the thoracic cavity, resulting in an increased risk of poor recovery and postsurgical complications. The authors sought to refine this surgery to reduce animal pain and distress without compromising overall efficiency of the technique. Using a minimally invasive method that does not expose the thoracic cavity, the authors gave accurately placed intrathymic injections, as confirmed by analyses with a reporter dye. They describe this new approach for intrathymic injection in mice that reduces complications associated with lengthy periods of anesthesia and thoracic cavity exposure.     

Pulmonary hypoplasia in chondrodystrophic mice

Lungs of day-18 fetal mice with hereditary chondrodysplasia (cho) were examined histologically and biochemically for pulmonary hypoplasia. Compared with normal littermate controls, the mutant's lungs were smaller by 37 (wet weight) and 22% (dry weight). Total DNA and protein per whole lung were decreased by 13 and 19%, respectively. The significantly smaller-than-normal terminal sacs observed in histological sections of the mutant's lungs corresponded with the greater difference (37%) in lung wet weight. The developmental mechanism for this disorder was further explored by examining the volumes of thoracic cavity and amniotic fluid. The volume of the thoracic cavity of newborn mutants was less than half that of controls, suggesting that the pathogenetic mechanism for the hypoplastic lungs in chondrodysplastic mice includes thoracic dystrophy. Measurement of the amniotic fluid volume revealed polyhydramnios in the mutant, thereby ruling out oligohydramnios as a mechanism. The relevance of this study to human pulmonary hypoplasia in short-limb chondrodystrophy is discussed.     

Circulating T and B lymphocytes of the mouse. II. Lifespan

The average lifespan of circulating lymphocytes was investigated by determining the percentage of labeling of thoracic duct lymphocytes (TDL⁵) from mice injected with tritiated thymidine (³HT) for various periods. Percentage of labeling of TDL from normal CBA mice, which consist of approximately 85% T cells and 15% B cells, was found to be directly proportional to the time of ³HT administration. This technique thus failed to demonstrate the presence of more than one population of lymphocytes. Less than 50% of TDL were labeled after ³HT injection for 8 weeks.Percentage of labeling of TDL from nude mice (which consist solely of B cells) was likewise found to be directly proportional to the duration of ³HT injection but occurred at a rate three to four times faster than in non-T cell-depleted CBA mice. Further experiments, in which a marker for B cells was used, allowed the rate of ³HT labeling of B cells to be studied in normal CBA mice. These data corroborated the findings in nude mice and indicated that, with regard to lifespan, thoracic duct B cells consisted of a single population with an average lifespan of 5–7 weeks. Similarly it was calculated that the average lifespan of thoracic duct T cells was in the order of 4–6 months.Studies on the rate of formation of TDL during prolonged thoracic duct drainage of normal CBA mice indicated that the percentage of newly formed cells increased rapidly after 24-hr drainage. The total numbers of newly formed cells, however, were found to remain relatively constant throughout the period of drainage investigated (up to 9 days) except for a transient increase during the second and third day. Newly formed small lymphocytes were found to consist of approximately equal proportions of T cells, B cells, and other “mononuclear” cells which lacked surface markers for either T or B cells. The great majority of large lymphocytes, in contrast, were found to be neither T cells nor B cells and probably belonged to the plasma cell line. In nude mice, production of newly formed lymphocytes during prolonged thoracic duct drainage was found to be very low in comparison with normal CBA mice.     

Infection in Mouse Peritoneal Cavity with a Pyrimidine-requiring Mutant and Naturally Occurring Staphylococcus aureus Strains

The lethal activity of a thymineless mutant of Staphylococcus aureus Wood 46 strain has been compared with that of three naturally occurring strains: parent Wood 46, Smith, and coagulase-negative SA-13. The thymineless mutant and the parent Wood 46 strain showed a sharp decline in culturable units from the peritoneal cavity in the first 4 hr after their injection. After 6 hr, that is, 2 hr before the mice began to die, the number of culturable units of the thymineless mutant was still declining, whereas that of the parent strain increased; for both strains, the number of units was still lower than that of the inoculum. Although the thymineless mutant, unlike the parent strain, was apparently unable to multiply in mouse peritoneal cavity, it killed mice at a similar rate. The highly virulent Smith strain known to multiply rapidly and the avirulent coagulase-negative SA-13 strain were used as additional controls. Under our experimental conditions, death of mice after the injection of the thymineless mutant in the peritoneal cavity did not seem to be due to bacterial multiplication but to toxicity, death being delayed by antitoxin. The pyrimidine-requiring auxotroph we used could be better material than killed bacteria to study some aspects of the lethal activity of S. aureus.     

Lack of interferon-gamma confers impaired neutrophil granulocyte function and imparts prolonged survival of adult filarial worms in murine filariasis

We investigated the role of IFN-gamma in host defense during murine filariasis. Using the fully permissive infection of BALB/c mice with the rodent filaria Litomosoides sigmodontis, we show that interferon (IFN)-gamma is essential for encapsulation of adult filarial worms in inflammatory nodules and for normal worm clearance. IFN-gamma knockout (KO) mice had only one third of the nodules of wild-type mice but displayed a more than twofold increase in worm burden and increased microfilaremia. Neutrophil granulocytes, but not macrophages or eosinophils, appear to directly control worm load and nodule formation. Neutrophils, which we showed earlier to be essential for the encapsulation process in the thoracic cavity, where the worms reside, were diminished at this location in IFN-gamma KO compared to wild-type mice; they also displayed strongly reduced chemotactic and phagocytic activity compared to neutrophils of controls. This argues for a distinct defect in neutrophil activation accounting for the low formation of inflammatory nodules. Tumor necrosis factor-alpha, a major neutrophil-activating cytokine expressed by macrophages in the thoracic cavity around the worms, was highly induced in wild-type but absent in KO mice. Diminished activation of neutrophils seems to be a general hallmark of IFN-gamma KO mice, since neutrophils from uninfected KO mice also showed a reduction in chemotactic and phagocytic activity when induced by casein. In conclusion, these data are the first to define an IFN-gamma-dependent immune effector mechanism in murine filarial infection, i.e. neutrophil-mediated control of the adult worm load.     

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.     

A chronic inflammatory response. Its role in supporting the development of c-myb and c-myc related promonocytic and monocytic tumors in BALB/c mice

This study demonstrates that an inflammatory response caused by the injection of pristane into the peritoneal cavity of mice provides a useful system for rapid induction of myeloid tumors by retroviruses. Two such tumors, which developed in the peritoneal cavity with average latencies of 68 to 71 d and incidences of greater than 50%, are 1) the McML, mature monocyte-macrophage tumors induced by retroviral constructs containing exons 2 and 3 of c-myc cDNA, and 2) the MML, promonocytic tumors induced by Moloney murine leukemia virus infection and its integration into the c-myb locus. Development of both neoplasms is clearly dependent on the intense i.p. inflammatory response, inasmuch as mice given the viruses and not pristane fail to develop these tumors. Although both types of tumors appear in the peritoneal cavity, the MML tumors that arise by i.v. injection of Moloney murine leukemia virus may actually originate via infection, and perhaps transformation, of precursor hemopoietic cells outside the peritoneal cavity, followed by migration of the cells to the peritoneal cavity. This is suggested by the fact that i.v.v but not i.p. injection of virus is an efficient method of producing these particular myeloid tumors. Although both McML and MML tumors require the inflammatory environment for their development, treatment of mice with a nonsteroid anti-inflammatory drug, indomethacin, has no effect on McML monocyte/macrophage tumors but completely prevents the development of the MML promonocyte tumors.     

Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

Indocyanine Green Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal vasculature of various eye diseases such as age-related macular degeneration (AMD). ICGA is especially useful to image the posterior choroidal vasculature of the eye due to its capability of penetrating through the pigmented layer with its infrared spectrum. ICGA time course can be divided into early, middle, and late phases. The three phases provide valuable information on the pathology of eye problems. Although time-course ICGA by intravenous (IV) injection is widely used in the clinic for the diagnosis and management of choroid problems, ICGA by intraperitoneal injection (IP) is commonly used in animal research. Here we demonstrated the technique to obtain high-resolution ICGA time-course images in mice by tail-vein injection and confocal scanning laser ophthalmoscopy. We used this technique to image the choroidal lesions in a mouse model of age-related macular degeneration. Although it is much easier to introduce ICG to the mouse vasculature by IP, our data indicate that it is difficult to obtain reproducible ICGA time course images by IP-ICGA. In contrast, ICGA via tail vein injection provides high quality ICGA time-course images comparable to human studies. In addition, we showed that ICGA performed on albino mice gives clearer pictures of choroidal vessels than that performed on pigmented mice. We suggest that time-course IV-ICGA should become a standard practice in AMD research based on animal models.     

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.     

Antitumor effect of intrapleural administration of Lactobacillus casei in mice

Summary The antitumor effect of intrapleural (i.pl.) administration of Lactobacillus casei YIT 9018 (LC 9018) on Meth A sarcoma in BALB/c mice was examined. Inoculation of Meth A cells into the thoracic cavity of BALB/c mice caused growth of the cells and the mice died from the tumor with an increased amount of pleural fluid. LC 9018 was given i.pl. to BALB/c mice before or after i.pl. inoculation of Meth A cells and the survival of the mice was determined. The i.pl. administration of LC 9018 was effective in prolonging the survival of the mice after i.pl. inoculation of Meth A tumor, and pretreatment with LC 9018 i.pl. also prolonged survival. Moreover, i.pl. administration of LC 9018 not only increased the number of thoracic exudate cells (TEC) but also augmented both cytolytic activity of thoracic macrophages and natural killer cell activity of TEC. Furthermore, phagocytic activity of thoracic macrophages against sheep red blood cells was enhanced and Ia antigen-positive cells in TEC were increased by the i.pl. treatment with LC 9018. These results showed that TEC induced by i.pl. administration of LC 9018 had antitumor activity against Meth A tumor inoculated i.pl. into BALB/c mice.     

Litomosoides sigmodontis: dynamics of the survival of microfilariae in resistant and susceptible strains of mice

Litomosoides sigmodontis in the BALB/c mouse is the only model of filariasis which allows the observation of the complete development in an immunocompetent mouse. In this study, we injected microfilariae (mf) intravenously, as well as into the pleural cavity, the site of natural release of mf from adult female worms, and followed the kinetics of elimination within the host. In susceptible BALB/c mice, mf circulated at high levels in the blood. In contrast, in C57BL/6 mice, which are refractory to full development, mf were eliminated rapidly from the peripheral blood. However, 6 days after intrapleural injection, viable larvae could be found in the pleural cavity and lung capillaries of both susceptible and resistant strains. The numbers of mf in the pleural cavity and lung capillaries in individual mice were significantly correlated, but not dependent on strain or peripheral microfilaraemia. Thus, although C57BL/6 mice showed enhanced production of nitric oxide by pleural exudate cells and a faster change in the numbers of circulating leukocytes after injection, rapid killing of mf by cell or nitric oxide-mediated mechanisms were not the reason for the different outcome. Furthermore, 3 h after iv injection, only a small percentage of mf could be recovered from the peripheral circulation, indicating the presence of a reservoir for mf containment. In conclusion, injected mf showed disparate dynamics of persistence within susceptible and resistant hosts, which is similar to the disparate outcome of natural infections with L. sigmodontis. This difference became obvious within 1 day after injection. The lung capillary system plays obviously a crucial part in regulation of microfilaremia. Our model also provides a possible means to explain frequent cases of occult infections in human filariasis.     

A SERUM FACTOR INDUCING MONOCYTOSIS DURING AN ACUTE INFLAMMATORY REACTION CAUSED BY NEWBORN CALF SERUM

An intraperitoneal injection of newborn calf serum (NBCS) into CRF Swiss mice causes an inflammatory reaction characterized by an increase in the number of macrophages in the peritoneal cavity and a concomitant monocytosis. The serum of such mice contains a monocytosis-inducing factor, as demonstrated by the intravenous injection of serum collected 18 (CalS 18) and 24 hr (CalS24) after the intraperitoneal injection of NBCS. Serum from normal untreated mice, from mice given an intraperitoneal injection of sterile pyrogen-free saline, which does not cause an inflammatory reaction, or from mice 72 hr after an intraperitoneal injection of NBCS, when the inflammatory reaction has subsided, does not cause a monocytosis in test mice. Intravenous injection of CalS 18 causes not only a monocytosis but also an increase in the number of promonocytes and bone marrow monocytes, suggesting an increased production of monocytes. The effect of CalS 18, CalS24 and CalS 18 filtrate is specific for the mononuclear phagocytes, since only non-significant increases in the numbers of lymphocytes and granulocytes were observed. The active factor in CalS 18 was shown to be different from the monocytosis-inducing factor present in NBCS. The monocytosis-inducing factor in CalS 18 passes through an ultrafiltration membrane with an exclusion limit of 50,000 Daltons, so that the molecular weight must be below this value.     

Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice

Osmotic pumps continuously deliver compounds at a constant rate into small animals. This article introduces a standard protocol used to induce aortic aneurysms via subcutaneous infusion of angiotensin II (AngII) from implanted osmotic pumps. This protocol includes calculation of AngII amount and dissolution, osmotic pump filling, implantation of osmotic pumps subcutaneously, observation after pump implantation, and harvest of aortas to visualize aortic aneurysms in mice. Subcutaneous infusion of AngII through osmotic pumps following this protocol is a reliable and reproducible technique to induce both abdominal and thoracic aortic aneurysms in mice. Infusion durations range from a few days to several months based on the purpose of the study. AngII 1,000 ng/kg/min is sufficient to provide maximal effects on abdominal aortic aneurysmal formation in male hypercholesterolemic mouse models such as apolipoprotein E deficient or low-density lipoprotein receptor deficient mice. Incidence of abdominal aortic aneurysms induced by AngII infusion via osmotic pumps is 5 - 10 times lower in female hypercholesterolemic mice and also lower in both genders of normocholesterolemic mice. In contrast, AngII-induced thoracic aortic aneurysms in mice are not hypercholesterolemia or gender-dependent. Importantly, multiple features of this mouse model recapitulate those of human aortic aneurysms.     

Migration of natural suppressor cells from bone marrow to peritoneal cavity by live BCG

Delayed-type hypersensitivity (DTH) responses were suppressed in mice inoculated with bone marrow cells from mice that had been injected with 10(8) colony-forming units (CFU) of live BCG. Upon analysis of this DTH-suppression by the use of a macrophage migration inhibition (MI) assay, the in vitro correlate of DTH, suppressor macrophages in the peritoneal cavity were found to play an important role in DTH suppression. However, neither suppression of DTH nor production of suppressor macrophages was observed in mice inoculated with bone marrow cells from mice that had been injected with methotrexate (MTX), a folic acid antagonist, and 10(8) CFU of live BCG. Moreover, suppressor cells against the MI activity of peritoneal exudate cells from BCG cell wall-immunized mice existed in bone marrow cells from normal mice, natural suppressor (NS) cells, and they were sensitive to MTX. In addition, these NS cells phagocytized carbonyl iron particles, were adherent to Sephadex G-10, and had Fc receptors, but they had no B or T cell markers, suggesting that these cells belonged to a macrophage compartment. From this evidence, we hypothesized that the origin of suppressor macrophages in the peritoneal cavity induced by live BCG injection was MTX-sensitive NS cells in bone marrow, and that these NS cells were stimulated by a small dose of live BCG trapped in bone marrow after i.v. injection of a high dose of live BCG and migrated from bone marrow to the peritoneal cavity.     

Deletion of IFT80 Impairs Epiphyseal and Articular Cartilage Formation Due to Disruption of Chondrocyte Differentiation

Intraflagellar transport proteins (IFT) play important roles in cilia formation and organ development. Partial loss of IFT80 function leads Jeune asphyxiating thoracic dystrophy (JATD) or short-rib polydactyly (SRP) syndrome type III, displaying narrow thoracic cavity and multiple cartilage anomalies. However, it is unknown how IFT80 regulates cartilage formation. To define the role and mechanism of IFT80 in chondrocyte function and cartilage formation, we generated a Col2α1; IFT80^f/f mouse model by crossing IFT80^f/f mice with inducible Col2α1-CreER mice, and deleted IFT80 in chondrocyte lineage by injection of tamoxifen into the mice in embryonic or postnatal stage. Loss of IFT80 in the embryonic stage resulted in short limbs at birth. Histological studies showed that IFT80-deficient mice have shortened cartilage with marked changes in cellular morphology and organization in the resting, proliferative, pre-hypertrophic, and hypertrophic zones. Moreover, deletion of IFT80 in the postnatal stage led to mouse stunted growth with shortened growth plate but thickened articular cartilage. Defects of ciliogenesis were found in the cartilage of IFT80-deficient mice and primary IFT80-deficient chondrocytes. Further study showed that chondrogenic differentiation was significantly inhibited in IFT80-deficient mice due to reduced hedgehog (Hh) signaling and increased Wnt signaling activities. These findings demonstrate that loss of IFT80 blocks chondrocyte differentiation by disruption of ciliogenesis and alteration of Hh and Wnt signaling transduction, which in turn alters epiphyseal and articular cartilage formation.     

Further studies on amplification of cell-associated immunological memory by secondary antigenic stimulus.

Using the hapten-carrier system in which the dinitrophenyl group (DNP) served as a B cell reactive hapten and bovine serum albumin (BSA) or human gammaglobulin (HGG) as a T cell reactive carrier, changes in the hapten-specific memory (B cell-associated memory) and the carrier-specific memory (T cell-associated memory) after a secondary antigenic stimulus were analyzed in mice. Since an immunological adjuvant was indispensable in the induction of the primary increase in memory, antigen used for the primary antigenic stimulus was injected together with the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) which has already been shown to exhibit a potent adjuvant effect. With the cell-transfer technique, it was found that the cell-associated hapten-specific memory for anti-DNP antibody response to DNP-BSA was truly amplified by the secondary injection of DNP-HGG into mice primed with DNP-HGG, and that the cell-associated carrier-specific memory as judged by the helper effect on anti-DNP response to DNP-BSA was also truly amplified by the secondary injection of BSA into mice primed with BSA. However, when memory was assessed in actively immunized mice, the secondary injection of BSA into mice primed with DNP-BSA and HGG decreased anti-DNP responsiveness to the tertiary injection of DNP-BSA, whereas the secondary injection of DNP-HGG secondarily increased anti-DNP responsiveness. In mice primed with DNP-BSA the titers of serum antibodies to BSA increased after the secondary injection of DNP-BSA or BSA. From these results it has been concluded that, like B cell-associated memory, T cell-associated memory is also amplified by a secondary antigenic stimulus, although its expression is inhibited in actively immunized mice through negative control by their antibodies.     

Nitric Oxide Release from the Liver Surface to the Intraabdominal Cavity during Acute Endotoxemia in Rats

Nitric oxide (NO) is produced by the liver during lipopolysaccharide (LPS)-induced endotoxemia. The aim of this study was to examine whether NO, which is produced in the liver, is released from the liver surface to the intraabdominal cavity during endotoxemia. NO was quantitatively determined by chemiluminescence and a newly developed gas purge technique was used to directly measure NO released from the liver surface and the intraabdominal cavity of rats before and after LPS (0.1 mg/kg, intraperitoneally) or saline administration. The expression of inducible NO synthase (iNOS) mRNA in the liver was detected by Northern blot analysis. NO levels from both the liver surface and in the intraabdominal cavity were elevated at 2 h after LPS injection and peaked at 10 h and both the time course of NO level were well correlated with each other. Both NO levels were below the detectable range before LPS and after saline administration. Inducible NOS mRNA in the liver exhibited a sharp increase to a maximum level at 4 h after LPS injection. The present study indicates that the hepatic NO, which might have been produced by iNOS in the liver, is released from the liver surface to the intraabdominal cavity during endotoxemia.     

A serum facted by newborn calf serum.

An intraperitoneal injection of newborn calf serum (NBCS) into CRF Swiss mice causes an inflammatory reaction characterized by an increase in the number of macrophages in the peritoneal cavity and a concomitant monocytosis. The serum of such mice contains a monocytosis-inducing factor, as demonstrated by the intravenous injection of serum collected 18 (CalS18) and 24 hr (CalS24) after the intraperitoneal injection of NBCS. Serum from normal untreated mice, from mice given an intraperitoneal injection of sterile pyrogen-free saline, which does not cause an inflammatory reaction, or from mice 72 hr after an intraperitoneal injection of NBCS, when the inflammatory reaction has subsided, does not cause a monocytosis in test mice. Intravenous injection of CalS18 causes not only a monocytosis but also an increase in the number of promonocytes and bone marrow monocytes, suggesting an increased in the number of promonocytes and bone marrow monocytes, suggesting an increased production of monocytes. The effect of CalS18, CalS24 and CalS18 filtrate is specific for the mononuclear phagocytes, since only non-significant increases in the numbers of lymphocytes and granulocytes were observed. The active factor in CalS18 was shown to be different from the monocytosis-inducing factor present in NBCS. The monocytosis-inducing factor in CalS18 passes through an ultrafiltration membrane with an exclusion limit of 50,000 Daltons, so that the molecular weight must be below this value.     

Comparison of the lateral tail vein and the retro-orbital venous sinus as routes of intravenous drug delivery in a transgenic mouse model

In mice, intravenous injections are commonly administered in the lateral tail vein. This technique is sometimes difficult to carry out and may cause stress to mice. Though injection through the retro-orbital venous sinus can provide certain advantages over lateral tail vein injection, this method is poorly defined and infrequently used. To compare the efficacy of these two routes of drug delivery, the authors injected MAFIA transgenic mice with the depletion agent AP20187, which selectively induces apoptosis in macrophages. Each mouse received five consecutive daily injections through either the lateral tail vein or the retro-orbital venous sinus. The authors then compared macrophage depletion in different tissues (lung, spleen, bone marrow and peritoneal exudate cells). Both routes of injection were similarly effective. A separate experiment using BALB/c mice indicated that retro-orbital venous sinus injection was the less stressful of the two methods.