Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.     

Manipulation of life-history decisions using leptin in a wild passerine

Seasonal timing of reproduction and the number of clutches produced per season are two key avian life-history traits with major fitness consequences. Female condition may play an important role in these decisions. In mammals, body condition and leptin levels are correlated. In birds, the role of leptin remains unclear. We did two experiments where we implanted female great tits with a pellet releasing leptin evenly for 14 days, to manipulate their perceived body condition, or a placebo pellet. In the first experiment where females were implanted when feeding their first brood offspring we found, surprisingly, that placebo treated females were more likely to initiate a second brood compared to leptin treated females. Only one second brood fledged two chicks while five were deserted late in the incubation stage or when the first egg hatched. No difference was found in female or male return rate or in recruitment rate of fledglings of the first brood, possibly due to the desertion of the second broods. In our study population, where there is selection for early egg laying, earlier timing of reproduction might be hampered by food availability and thus nutritional state of the female before egg laying. We therefore implanted similar leptin pellets three weeks before the expected start of egg laying in an attempt to manipulate the laying dates of first clutches. However, leptin treated females did not initiate egg laying earlier compared to placebo treated females, suggesting that other variables than the perceived body condition play a major role in the timing of reproduction. Also, leptin treatment did not affect body mass, basal metabolic rate or feeding rates in captive females. Manipulating life history decisions using experimental protocols which do not alter individuals' energy balance are crucial in understanding the trade-off between costs and benefits of life history decisions.     

Pituitary adenylate cyclase-activating polypeptide maintains neonatal breathing but not metabolism during mild reductions in ambient temperature

Mild reductions in ambient temperature dramatically increase the mortality of neonatal mice deficient in pituitary adenylate cyclase-activating polypeptide (PACAP), with the majority of animals succumbing in the second postnatal week. During anesthesia-induced hypothermia, PACAP(-/-) mice at this age are also vulnerable to prolonged apneas and sudden death. From these observations, we hypothesized that before the onset of genotype-specific mortality and in the absence of anesthetic, the breathing of PACAP-deficient mice is more susceptible to mild reductions in ambient temperature than wild-type littermates. To test this hypothesis, we recorded breathing in one group of postnatal day 4 PACAP+/+, (+/-), and (-/-) neonates (using unrestrained, flow-through plethysmography) and metabolic rate in a separate group (using indirect calorimetry), both of which were exposed acutely to ambient temperatures slightly below (29 degrees C), slightly above (36 degrees C), or at thermoneutrality (32 degrees C). At 32 degrees C, the breathing frequency of PACAP(-/-) neonates was significantly less than PACAP+/+ littermates. Reducing the ambient temperature to 29 degrees C caused a significant suppression of tidal volume and ventilation in both PACAP+/- and (-/-) animals, while the tidal volume and ventilation of PACAP+/+ animals remained unchanged. Genotype had no effect on the ventilatory responses to ambient warming. At all three ambient temperatures, genotype had no influence on oxygen consumption or body temperature. These results suggest that during mild reductions in ambient temperature, PACAP is vital for the preservation of neonatal tidal volume and ventilation, but not for metabolic rate or body temperature.     

TESTING THE QUALITY OF A CARRIER: A FIELD EXPERIMENT ON LIZARD SIGNALERS

In the Australian painted dragon lizard ( Ctenophorus pictus ), males occur in two different morphs with respect to gular color, with or without a yellow bib. Males without a bib lost within-clutch paternity significantly more often to rivals than bibbed males. Thus, it appears that bibs identify some phenotypic advantage linked to competitive ability. To test whether this could be related to whole-organism capacity to withstand an increased workload (due to better health and vigor, or evolved differences in self-maintenance), we implanted males with a lead pellet (loaded), Styrofoam pellet (controls), or sham-operated males without implants (shams), and compared male categories with respect to how they maintained body mass during the mating season. Somewhat unexpectedly, bibbed males consistently lost more body weight across all treatments and controls, although we could not verify that this translated into higher mortality in this short-lived animal (about 80% survive for one year only). However, bibbed males may invest more into "mating success" than nonbibbed males, which agrees with our experimental results and paternity data.     

Comparison of two methods (left carotid artery and abdominal aorta) for surgical implantation of radiotelemetry devices in CD-1 mice

The goal of this study was to compare two surgical methods, the left carotid (LC) and the abdominal aorta (AA), for mouse instrumentation with telemetry devices, to determine the best method for measuring cardiovascular (CV) parameters by radiotelemetry in freely moving mice. Surgery success rate, postsurgical recovery rate, clinical parameters, CV data (baseline and response to nicotine) and circadian rhythm measurements were compared between these techniques. Brains of LC-implanted mice were evaluated for potential ischaemia by direct observation of the Circle of Willis anatomy and histopathology. For this purpose, a total of 31 CD-1 male mice were instrumented with PA C20 devices (10 with LC and 21 with AA). Mortality, morbidity, physical examination, body weight (BW), water and food consumption (W/FC), mean blood pressure (MBP) and heart rate (HR) were monitored daily during the recovery period (10 days). CV baseline data were recorded continuously during two periods of four days, and finally, both LC- and AA-implanted mice received an acute subcutaneous administration of 1 mg/kg nicotine; BP and HR were recorded during 5 h after nicotine administration. Results showed that, in LC-implanted mice, 80% survived surgery and recovered well. In contrast, only 57% of mice implanted with the AA technique survived surgery and some presented lethal complications. Both techniques had similar recovery times for BW and W/FC, comparable return to normal circadian rhythm (day 6 post-surgery) and similar CV baseline values. No significant differences were observed in CV response to nicotine between both groups of implanted CD-1 mice. No histopathological changes suggestive of ischaemia were noted in the brain of mice implanted in the LC. Six out of the eight LC-implanted mice remained in good health and had good pressure signal for at least 100 days post-surgery, while most of the AA-implanted mice lost the signal pressure within 14-49 days post-surgery. In conclusion, we believe that LC implantation in mice is superior to the AA technique and is more appropriate for long-term telemetry studies, especially for smaller (transgenic) animals.     

Development and Verification of a Mouse Model for Roux-en-Y Gastric Bypass Surgery with a Small Gastric Pouch

Existing mouse models of Roux-en-Y gastric bypass (RYGB) surgery are not comparable to human RYGB in gastric pouch volume for a large or absent gastric volume. The aim of this study was to develop and characterize a mouse RYGB model that closely replicates gastric pouch size of human RYGB surgery of about 5% of total gastric volume. We established this model in diet-induced obese (DIO) mice of C57BL/6J. This surgery resulted in a sustained 30% weight loss, entirely accounted for by decreased fat mass but not lean mass, compared to sham-operated mice on the high fat diet. Compared to sham-operated mice, energy expenditure corrected for total body weight was significantly increased by about 25%, and substrate utilization was shifted toward higher carbohydrate utilization at 8 weeks after RYGB when body weight had stabilized at the lower level. The energy expenditure persisted and carbohydrate utilization was even more pronounced when the mice were fed chow diet. Although significantly increased during daytime, overall locomotor activity was not significantly different. In response to cold exposure, RYGB mice exhibited an improved capacity to maintain the body temperature. In insulin tolerance test, exogenous insulin-induced suppression of plasma glucose levels was significantly greater in RYGB mice at 4 weeks after surgery. Paradoxically, food intake measured at 5 weeks after surgery was significantly increased, possibly in compensation for increased fecal energy loss and energy expenditure. In conclusion, this new model is a viable alternative to existing murine RYGB models and the model matches human RYGB surgery in anatomy. This model will be useful for studying molecular mechanisms involved in the beneficial effects of RYGB on body weight and glucose homeostasis.     

Biocompatibility and long-term toxicity of InnoPol implant, a biodegradable polymer scaffold.

InnoPol, a poly((D,L)-lactic-co-glycolic acid) [PLGA] 65/35 scaffold manufactured by special gas foaming methods in Korea, was subjected to tests to evaluate the degradation and tissue compatibility characteristics and long-term systemic toxicity in mice and rats. C57BL/6 mice and SD rats were implanted subcutaneously with 3-mm- and 1-mm-thick InnoPol circular discs, 10 mm in diameter, respectively, and sacrificed 8, 12, and 24 weeks after implantation. No test material-related effects were observed in mortality, clinical signs, body weight gain, food and water consumption, ophthalmologic signs, urinalysis, hematology, serum biochemistry parameters and organ weights of all animals implanted with InnoPol. Also, there were no systemic symptoms including metabolic alterations and inflammatory reactions in either mice or rats. In addition, no gross pathological findings, except skin lesions around the implantation sites, were found in the major organs. Although mild inflammation at the site of InnoPol implantation was confirmed from hematoxylin and eosin or Masson's trichrome staining at 8-12 weeks, the reactions had disappeared at 24 weeks following complete degradation of the scaffold, leaving granulomatous tissues that were similar to surgical wounds in sham operation controls without implants. These results suggest that InnoPol possesses good mechanical properties and tissue compatibility and does not cause any systemic toxicity other than transient local inflammatory reactions at the implantation site, and that it might be useful in applications as a medical device for implantation.     

Chronic measurement of cardiac output in conscious mice

We describe the feasibility of chronic measurement of cardiac output (CO) in conscious mice. With the use of gas anesthesia, mice >30 g body wt were instrumented either with transit-time flow probes or electromagnetic probes placed on the ascending aorta. Ascending aortic flow values were recorded 6-16 days after surgery when probes had fully grown in. In the first set of experiments, while mice were under ketamine-xylazine anesthesia, estimates of stroke volume (SV) obtained by the transit-time technique were compared with those simultaneously obtained by echocardiography. Transit-time values of SV were similar to those obtained by echocardiography. The average difference +/- SD between the methods was 2 +/- 7 microl. In the second set of studies, transit-time values of CO were compared with those obtained by the electromagnetic flow probes. In conscious resting conditions, estimates +/- SD) of cardiac index (CI) obtained by the transit-time and electromagnetic flow probes were 484 +/- 119 and 531 +/- 103 ml x min(-1) x kg body wt(-1), respectively. Transit-time flow probes were also implanted in mice with a myocardial infarction (MI) induced by ligation of a coronary artery 3 wk before probe implantation. In these MI mice (n = 7), average (+/- SD) resting and stimulated (by volume loading) values of CO were significantly lower than in noninfarcted mice (n = 15) (resting CO 16 +/- 3 vs. 20 +/- 4 ml/min; stimulated CO 20 +/- 5 vs. 26 +/- 6 ml/min). Finally, using transfer function analysis, we found that, in resting conditions for both intact and MI mice, spontaneous variations in CO (> 0.1 Hz) were mainly due to those occurring in SV rather than in heart rate. These data indicate that CO can be measured chronically and reliably in conscious mice, also in conditions of heart failure, and that variations in preload are an important determinant of CO in this species.     

Analgesic effects of tramadol, carprofen or multimodal analgesia in rats undergoing ventral laparotomy

In this study, the authors evaluated the analgesic efficacy of tramadol (an opioid-like analgesic), carprofen (a nonsteroidal anti-inflammatory drug) and a combination of both drugs (multimodal therapy) in a rat laparotomy model. The authors randomly assigned rats to undergo either surgery (abdominal laparotomy with visceral manipulation and anesthesia) or anesthesia only. Rats in each group were treated with tramadol (12.5 mg per kg body weight), carprofen (5 mg per kg body weight), a combination of tramadol and carprofen (12.5 mg per kg body weight and 5 mg per kg body weight, respectively) or saline (anesthesia control group only; 5 mg per kg body weight). The authors administered analgesia 10 min before anesthesia, 4 h after surgery or (for the rats that received anesthesia only) anesthesia and 24 h after surgery or anesthesia. They measured locomotor activity, running wheel activity, feed and water consumption, body weight and fecal corticosterone concentration of each animal before and after surgery. Clinical observations were made after surgery or anesthesia to evaluate signs of pain and distress. The authors found that carprofen, tramadol and a combination of carprofen and tramadol were all acceptable analgesia regimens for a rat laparotomy model.     

Conditionally immortalised neural stem cells promote functional recovery and brain plasticity after transient focal cerebral ischaemia in mice

Cell therapy has enormous potential to restore neurological function after stroke. The present study investigated effects of conditionally immortalised neural stem cells (ciNSCs), the Maudsley hippocampal murine neural stem cell line clone 36 (MHP36), on sensorimotor and histological outcome in mice subjected to transient middle cerebral artery occlusion (MCAO).Adult male C57BL/6 mice underwent MCAO by intraluminal thread or sham surgery and MHP36 cells or vehicle were implanted into ipsilateral cortex and caudate 2 days later. Functional recovery was assessed for 28 days using cylinder and ladder rung tests and tissue analysed for plasticity, differentiation and infarct size.MHP36-implanted animals showed accelerated and augmented functional recovery and an increase in neurons (MAP-2), synaptic plasticity (synaptophysin) and axonal projections (GAP-43) but no difference in astrocytes (GFAP), oligodendrocytes (CNPase), microglia (IBA-1) or lesion volumes when compared to vehicle group.This is the first study showing a potential functional benefit of the ciNSCs, MHP36, after focal MCAO in mice, which is probably mediated by promoting neuronal differentiation, synaptic plasticity and axonal projections and opens up opportunities for future exploitation of genetically altered mice for dissection of mechanisms of stem cell based therapy.     

Impact of 'living apart together' on postoperative recovery of mice compared with social and individual housing

Social housing is the optimal way of housing female laboratory mice. However, individual housing may be required in experimental designs, for example after surgery. We therefore investigated whether housing two female mice in a cage, separated by a grid partition ('living apart together', LAT), counters the adverse effects of individual housing on postoperative recovery. Ten individually housed (IND) mice, nine socially housed (SOC) mice and nine mice, housed LAT, were surgically implanted with a telemetry transmitter. From one week prior to surgery until three weeks thereafter, several physiological and behavioural parameters were measured in the mice subjected to surgery. The telemetry transmitter measured heart rate (HR), body temperature and activity continuously. Body weight, food and water intake were scored regularly, as were wound healing, ease of handling, nest building and behaviour. Results indicated that SOC mice appear to be less affected by abdominal surgery than IND mice, as indicated by HR and behaviour. LAT, however, did not appear to be beneficiary to the mice. Increased HR levels and differences in behaviour as compared with both SOC and IND animals indicate that LAT may even be the most stressful of the three housing conditions. We therefore conclude that mice benefit most from social housing after surgery. If, however, social housing is not possible, individual housing appears to be a better option than separating mice by a grid partition.     

Evaluation of ethylene-vinyl acetate copolymer as a non-inflammatory alternative to Freund's complete adjuvant in rabbits

Ethylene-vinyl acetate copolymer (EVAc) was evaluated as an antigen delivery device in laboratory rabbits. Bovine serum albumin (BSA) was incorporated with EVAc in a pellet, which was implanted subcutaneously. Serum antibody titers to BSA in four implanted rabbits were equal to titers in four rabbits injected twice with BSA in complete Freund's adjuvant. Three of four rabbits implanted with EVAc displayed no inflammation or systemic illness in response to the pellet. The fourth rabbit repeatedly developed a small abscess at the implantation site, but the lesions were less severe than complete Freund's adjuvant injection sites. The EVAc pellet is recommended as a non-inflammatory alternative method to Freund's adjuvants for producing serum antibody in rabbits.     

Effect of thermal environment upon transplanted tumors and growth of mice.

Ehrlich ascites tumor cells were inoculated subcutaneously in the dorsal, interscapular region of three groups of mice that were subsequently maintained in normo-, hypo- or hyperthermic environments. The controls, mice that had homologous whole spleens or thymic glands implanted in the same location, were maintained in a normothermic environment. Number of tumors established, their weights and volumes, and weights of animals were compared as well as tumor temperature, which was compared to brown fat and core temperatures.(1) The percentage of animals developing tumors was independent of environment. (2) Mice with tumors, and mice in which tumors did not take, gained more weight at the normothermic temperatures (21 °C) than at either 31 or 5 °C. (3). When the weight and volume of tumors are expressed per gram of mouse body weight, there is no difference between the hypo- and normothermic environments, but the weights and volume of tumors in a hyperthermic environment are only 36 and 58% respectively of the tumors in normothermic animals. (4) Tumor temperatures are about 1 °C cooler than the underlying brown fat regardless of environment, and brown fat was 0.5 °C cooler than the core temperature. (5) Transplanted thymus glands and spleens become encysted after 31 days; they proved to be cooler than the malignant tumors.     

Flying distance of frass kicked by the grasshopper Atractomorpha lata and factors affecting the flying distance

Adults of the grasshopper Atractomorpha lata use a hind leg kick to project their frass a considerable distance from themselves. To clarify the defecation behavior quantitatively and collect basic information that aids in clarification of the adaptive significance of this behavior, we measured the flying distance of kicked frass and examined the factors affecting the flying distance in adult A. lata. Males and females kicked their frass an average of 252 and 487 mm away, respectively. This represented more than ten times the body length or 100 times the length of the frass pellet for either sex. Only sex affected the flying distance of frass. There were sexual differences in hind‐femur length (females longer than males), volume of frass pellet (females larger than males) and ratio of diameter to length of frass pellet (RFP; larger in males than in females). The flying distance appears to be affected by the femur length, volume of frass pellet and RFP when data of both males and females were combined for analysis. However, none of these effects were observed when testing the effects within each sex. These results suggest that the sex difference in the flying distance does not result from the sex difference in femur length, volume of frass pellet or RFP. Because A. lata kicked their frass far away in both sexes, the frass‐kicking behavior might give benefit common to both males and females.     

Role of hypothermia induced by tumor necrosis factor on apoptosis and function of inflammatory neutrophils in mice

Changes in body temperature and cell infiltration, mediated by cytokines including tumor necrosis factor-alpha (TNF-alpha), occur during inflammation, but a role of body temperature on inflammatory responses remains obscure. Intraperitoneal injection of 10% casein to mice resulted in transient hypothermia followed by neutrophil accumulation in peritoneal cavities. Peritoneal TNF-alpha was rapidly raised, and pretreatment of mice with an anti-TNF-alpha antibody promoted temperature restoration and partially inhibited neutrophil accumulation. To investigate direct effects of body temperature on neutrophils, peritoneal or peripheral blood neutrophils were cultured at 35 degrees C or 37 degrees C with or without recombinant murine TNF-alpha (100 ng/ml) or a protein synthesis inhibitor cycloheximide (1 microg/ml). Significant inhibition of spontaneous and TNF-alpha-induced apoptosis was obtained at 35 degrees C compared with 37 degrees C, an effect that was not altered by the addition of cycloheximide. Moreover, phagocytic ability of peritoneal neutrophils was significantly enhanced by incubating them at the lower temperature. These results indicate that mild hypothermia induced by endogenous TNF-alpha has enhancing roles on neutrophil survival and function during peritoneal inflammation.     

In vivo cellular uptake of bismuth ions from shotgun pellets

Shotgun pellets containing bismuth (Bi) are widely used and may cause a rather intense exposure of some wild animals to Bi. A Bi shotgun pellet was implanted intramuscularly in the triceps surae muscle of 18 adult male Wistar rats. Another group of 9 animals had a Bi shotgun pellet implanted intracranially in the neocortex. Eight weeks to 12 months later the release of Bi ions was analysed by autometallography (AMG) of tissue sections from different organs (brain, spinal cord, kidney, liver, testes). In the group with intramuscular Bi shotgun pellets no AMG staining could be found for the first 2-4 months; 6 months after exposure Bi was traced in the kidney. Twelve months after the implantation the kidneys were heavily loaded and Bi was also traced in testosterone-producing Leydig cells, in glial cells and in neurons of brain and spinal cord. In the central nervous system (CNS) motor neurons were the most loaded. In rats with intracranially implanted shotgun pellets a massive uptake of Bi was observed involving both glia and neurons throughout the brain. The cells close to the shotgun pellet had the highest uptake. The animals showed a pronounced Bi uptake in the ependyma cells lining the ventricular system and in the cubic epithelia covering the choroid plexus. Dissemination of Bi ions to the rest of the body was demonstrated by AMG tracing of Bi accumulations in the tubular cells of the kidney. These findings emphasize that metallic Bi, including shotgun pellets, represents sites of intense Bi pollution if implanted or shot into a living organism, and further that such metallic Bi bodies, if they enter the CNS, cause a spread of Bi ions throughout it.     

Continuous exposure of mice to superantigenic toxins induces a high-level protracted expansion and an immunological memory in the toxin-reactive CD4+ T cells

We analyzed the responses of several T cell fractions reactive with superantigenic toxins (SAGTs), staphylococcal enterotoxin A (SEA), or Yersinia pseudotuberculosis-derived mitogen (YPM) in mice implanted with mini-osmotic pumps filled with SEA or YPM. In mice implanted with the SEA pump, SEA-reactive Vbeta3(+)CD4(+) T cells exhibited a high-level protracted expansion for 30 days, and SEA-reactive Vbeta11(+)CD4(+) T cells exhibited a low-level protracted expansion. SEA-reactive CD8(+) counterparts exhibited only a transient expansion. A similar difference in T cell expansion was also observed in YPM-reactive T cell fractions in mice implanted with the YPM pump. Vbeta3(+)CD4(+) and Vbeta11(+)CD4(+) T cells from mice implanted with the SEA pump exhibited cell divisions upon in vitro restimulation with SEA and expressed surface phenotypes as memory T cells. CD4(+) T cells from mice implanted with the SEA pump exhibited high IL-4 production upon in vitro restimulation with SEA, which was due to the enhanced capacity of the SEA-reactive CD4(+) T cells to produce IL-4. The findings in the present study indicate that, in mice implanted with a specific SAGT, the level of expansion of the SAGT-reactive CD4(+) T cell fractions varies widely depending on the TCR Vbeta elements expressed and that the reactive CD4(+) T cells acquire a capacity to raise a memory response. CD8(+) T cells are low responders to SAGTs.     

Evaluating postoperative analgesics in mice using telemetry

The study examined the efficacy of preemptive or postoperative analgesia on surgical pain in the mouse. Radiotelemetry transmitters were surgically implanted in 28 female ICR mice. A mock ova implantation surgery was then performed. Mice were treated with a single dose of buprenorphine or flunixin meglumine prior to or after surgery, three doses of buprenorphine, or were untreated. Heart rate, blood pressure, home cage activity, food and water consumption, and body weight were measured. The no-analgesia group showed no significant differences between any parameters collected prior to surgery and those collected at similar times during the day of surgery. Significant increases in mouse activity on the day of surgery occurred with all analgesic treatments, compared with pre-surgical activity. There were no consistent significant changes in any other telemetry parameter after treatment with analgesics compared with no analgesia. Food consumption and body weight the day after surgery were reduced significantly in the animals treated with three doses of buprenorphine compared with untreated mice and mice given a single dose of buprenorphine. We conclude that the mock ova implant procedure does not induce sufficient pain to cause alterations in heart rate and blood pressure in the mouse. Activity was significantly reduced in the first 6 h after surgery in mice without analgesia, compared with activity prior to surgery. There were no significant differences between pre-emptive and postoperative analgesia. Body weight and food and water consumption were poor measures of pain because analgesia alone affected these parameters.     

Subcutaneous injection,from birth,of epigallocatechin-3-gallate,a component of green tea,limits the onset of muscular dystrophy in <Emphasis Type="Italic">mdx</Emphasis> mice: a quantitative histological,immunohistochemical and electrophysiological study

Dystrophic muscles suffer from enhanced oxidative stress. We have investigated whether administration of an antioxidant, epigallocatechin-3-gallate (EGCG), a component of green tea, reduces their oxidative stress and pathophysiology in mdx mice, a mild phenotype model of human Duchenne-type muscular dystrophy. EGCG (5 mg/kg body weight in saline) was injected subcutaneously 4× a week into the backs of C57 normal and dystrophin-deficient mdx mice for 8 weeks after birth. Saline was injected into normal and mdx controls. EGCG had almost no observable effects on normal mice or on the body weights of mdx mice. In contrast, it produced the following improvements in the blood chemistry, muscle histology, and electrophysiology of the treated mdx mice. First, the activities of serum creatine kinase were reduced to normal levels. Second, the numbers of fluorescent lipofuscin granules per unit volume of soleus and diaphragm muscles were significantly decreased by about 50% compared to the numbers in the corresponding saline-treated controls. Third, in sections of diaphragm and soleus muscles, the relative area occupied by histologically normal muscle fibres increased significantly 1.5- to 2-fold whereas the relative areas of connective tissue and necrotic muscle fibres were substantially reduced. Fourth, the times for the maximum tetanic force of soleus muscles to fall by a half increased to almost normal values. Fifth, the amount of utrophin in diaphragm muscles increased significantly by 17%, partially compensating for the lack of dystrophin expression.     

Establishment of a Non-Invasive Semi-Quantitative Bioluminescent Imaging Method for Monitoring of an Orthotopic Esophageal Cancer Mouse Model

Orthotopic models of various types of tumors are widely used in anti-tumor therapeutic experiments in preclinical studies. However, there are few ways to appropriately monitor therapeutic effect in orthotopic tumor models, especially for tumors invisible from the outside. In this study we aimed to establish a non-invasive semi-quantitative bioluminescent imaging method of monitoring an orthotopic esophageal cancer mouse model. We confirmed that the TE8 esophageal cancer cell line implanted orthotopically into the abdominal esophagus of nu/nu mice (n = 5) developed not only a main tumor at the implanted site, but also local lymph node metastases and peritoneal disseminations within 6 weeks after inoculation. We established a TE8 cell line that stably expressed the firefly luciferase gene (TE8-Luc). We showed that TE8-Luc cells implanted subcutaneously into nu/nu mice (n = 5) grew over time until 5 weeks after inoculation. Tumor volume was strongly correlated with luminescent intensity emitted from the tumor, which was quantified using the IVIS imaging system. We then showed that TE8-Luc cells implanted orthotopically into the mouse abdominal esophagus (n = 8) also formed a tumor and that the luminescent intensity of such a tumor, as detected by IVIS, increased over time until 7 weeks after inoculation and was therefore likely to reflect tumor progression. We therefore propose that this orthotopic esophageal cancer model, monitored using the non-invasive semi-quantitative IVIS imaging system, will be useful for in vivo therapeutic experiments against esophageal cancer. This experimental setting is expected to contribute to the development of novel therapeutic technologies for esophageal cancer in preclinical studies.