Adult C3H Mice Research Articles

The Radioprotector GC4419 Enhances the Response of Head and Neck Squamous Cell Carcinoma Tumors Tumors to Ionizing Radiation Alone and with Radioimmune Therapy

Despite aggressive treatment modalities including surgery and chemoradiation therapy, ∼30% of patients with squamous cell carcinoma of the head and neck (HNSCC) will present with recurrent disease for which the median survival is less than one year. Recently, stereotactic body radiation therapy (SBRT) in combination with anti PD-1 based immune-oncology (IO) has emerged as an effective salvage therapy in the recurrent setting. In a recent randomized, placebo controlled, Phase II clinical trial for patients undergoing chemoradiation therapy for treatment of HNSCC, the penta-aza macrocyclic compound GC4419 (St. Louis, MO) demonstrated a statistically significant reduction in the duration, incidence, and severity of patients who developed severe oral mucositis (SOM), proving its clinical utility as a radioprotector. Recent work in our laboratory using pre-clinical animal models for non-small cell lung cancer (NSCLC) demonstrated that GC4419 does not protect NSCLC tumors from radiation, but enhances the radiation response when utilized specifically with high dose per fraction exposures alone or combined immune therapy. These two pieces of evidence led us to hypothesize that GC4419 would be effective in enhancing SBRT combined with anti-PD-1 therapy in the setting of recurrent HNSCC, reducing the rate of normal tissue complications (SOM) while also improving survival. The AT-84 HNSCC tumor cell line, syngenic to the C3H mouse strain, was selected for these studies. Subcutaneous AT-84 tumors were treated with differing biologically equivalent radiation fractionation schedules of 17 Gy x 1 fx, 10.24 Gy x 2 fxn, 5 Gy x 5 fxn, and 2 Gy x 14 fxn in combination with an α-PD-1 antibody (BioXcell) and GC4419. Tumor growth delay (TGD) was measured. A TCD50 experiment is underway. To assess normal tissue protection, the epithelial layer thickness on the tongues of adult female C3H mice was measured 12 days post a single dose of 12 or 17 Gy. GC4419 significantly enhanced radiation induced TGD as a single agent regardless of the fractionation schedule, the exception being the 2 Gy x 14 fxn group which grew too quickly to administer the full course of radiation. α-PD-1 therapy did not enhance the efficacy of radiation in any dose per fraction schedule, however the triple combination of radiation, GC4419, and α-PD-1 therapy demonstrated an increased efficacy over radiation and GC4419 alone. This triple combination effect was more effective at higher doses per fraction. Experiments for normal tissue damage are ongoing and will be reported. The ability of GC4419 to enhance the radioresponse of syngenic AT-84 tumors to radiation alone, and a further enhancement of that response when combined with α-PD-1 therapy suggests that GC4419 will be effective at improving outcomes in those patients being treated for recurrent disease using immune-SBRT.

Irradiated sporozoite vaccination induces sex-specific immune responses and protection against malaria in mice

In both preclinical animal studies and human clinical trials, adult females tend to develop greater adaptive immune responses than males following receipt of either viral or bacterial vaccines. While there is currently no approved malaria vaccine, several anti-sporozoite vaccines, including RTS,S/AS01 and attenuated sporozoite vaccines, are in development, but the impact of sex and age on their efficacy remains undefined. To examine sex differences in the efficacy of anti-sporozoite stage malaria vaccination, adult (10 weeks of age) or juvenile (11 days of age) male and female C3H mice were twice vaccinated with irradiated transgenic Plasmodium berghei sporozoites expressing the P. falciparum circumsporozoite (CSP) protein and 45 days post boost vaccination, mice were challenged with transgenic P. berghei via mosquito bite or intradermal challenge. Immunization with irradiated sporozoites resulted in greater protection against challenge in adult females, which was associated with greater anti-CSP antibody production and avidity, as well as greater hepatic, but not splenic, CD8+ T cell IFNƴ production in adult females than adult males. No sex differences in adaptive immune responses or protection were observed in mice vaccinated prior to puberty, suggesting a role for sex steroid hormones. Depletion of testosterone in males increased, whereas rescue of testosterone decreased, anti-CSP antibody production, the number of antigen-specific CD8+ T cells isolated from the liver, and protection following parasite challenge. Conversely, depletion of sex steroids in female mice did not alter vaccine-induced responses or protection following challenge. These data suggest that elevated testosterone concentrations in males reduce adaptive immunity and contribute to sex differences in malaria vaccine efficacy.

Reprogramming and Recapitulating T Cells in the Immature Immune Environment of the Neonate to Induce Transplant Tolerance

Introduction: Semi-allogeneic bone marrow and spleen cells (BMC/SC) induce transplant tolerance in neonatal mice whereas allogeneic BMC/SC cause lethal graft-versus-host disease (GVHD). To prevent aGVHD and induce transplant tolerance with allo-SC/BMC we reprogrammed donor/host CD4 T cells by co-stimulation blockade to preserve regulatory CD4 T cells while recapitulating depleted donor/host CD8 T cells. Methods: C3H (H-2k) neonatal mice were injected iv with total or depleted B6 (H-2b) GFP+ SC/BMC. CD8 T and CD49b NK cells were depleted from donor inocula using StemCell Technologies kits. In vivo neonatal host CD8 T cells were depleted (Mab 53–6.7) and donor/host CD4 T cells reprogrammed (CD154 Mab MR1). Trafficking and interactions of injected cells were monitored by microscopy. Tolerance induction was assessed by transplanting treated mice as adults with donor-type hearts. Results: Neonatal mice injected with GFP+ allo-SC/BMC developed aGVHD, with diarrhea, reduced growth and early death. GFP+ cells proliferated and spread throughout injected mice indicating systemic inflammation. Depletion of donor/host CD8 T cells together with co-stimulation blockade of donor/host CD4 T cells (CD154) resulted in GFP signal being reduced and restricted to lymphoid organs (day 6). High resolution microscopy showed donor T and B cells positioned respectively in PALS and follicular regions of host spleen; lack of Ki67 immunostaining indicated many donor cells were not proliferating. Donor DC were detected in host thymus (day 6) suggesting a possible role in central tolerance. Donor-type B6 hearts transplanted into neonatally-treated adult C3H mice (n=5) (CD8 depleted/CD4 co-stimulation blockade) all continued to beat at 100 days post-transplant, 3 grafts with maximal strength and 2 grafts with diminished strength. In untreated control mice (n=3) donor-type hearts stopped beating by day 10. H&E staining of a strongly beating B6 heart graft at 100 days showed undamaged cardiomyocytes with little if any cellular infiltrate and absence of macrophages and T cells by immunostaining. Conclusion: Reprograming CD4 T cells while depleting/recapitulating CD8 T cells in allo-SC/BMC treated neonates leads to 100% cardiac allograft survival at 100days; low beat scores in 2/5 grafts suggests further optimization is required. These findings provide insight into robust tolerance induction in neonates. This project was supported with funding from the Canadian Institutes for Health Research and the Gordon English family.

Dermal wounds alter tumor immunobiology in mice

Tumor growth, angiogenesis, and invasion are modulated by tumor-infiltrating myeloid cells. The production of these cells by the bone marrow is an energetically-costly process, and therefore, finite and tightly regulated. Both prophylactic (e.g., biopsy) and direct cancer treatment (e.g., tumor surgery) require often elaborate skin wound healing, another myeloid cell-recruiting process. Thus, we tested the hypothesis that dermal wound healing depletes tumors of some myeloid cell populations, thereby affecting tumor immunobiology, and ultimately, increases tumor growth. Oral cancer cells (106) or PBS were injected (s.c.) into the flank of adult female immunocompetent C3H mice. Sixteen days post-tumor induction, two 3.5 mm excisional wounds were placed through the dorsal skin; other mice remained unwounded. Blood, wounds, bone marrow, spleen, tumor, and tumor-draining lymph nodes were collected 1 or 5 days later. In mice that were wounded, tumor mass modestly increased five days after wounding. The numbers of circulating leukocytes were not significantly affected by wounding; however, tumors increased the percentages of neutrophils and monocytes and decreased lymphocytes. Tumor gene expression of various inflammatory markers (CCL2, CXC10, IL-6, Tnfalpha, MIP-1) was modulated by whether or not a wound was also present. Flow cytometry will determine immune cell trafficking throughout the body. The goal of this work is to provide an understanding of how standard clinical treatments affect tumor immunobiology, and therefore, tumor growth and metastases.

Effects of FK-506 and CTLA4–Ig on nerve allografts in mice

The purpose of this study is to evaluate the effects of FK-506 and cytotoxic T lymphocyte-associated antigen-4 immunoglobulin fusion protein (CTLA4--Ig) on nerve allografts. Adult male inbred C3H mice (mean weight, 25 g) surgically received 8 mm posterior tibial nerve defects, and donor nerve allografts from donor male C57BL mice were implanted. The experimental animals were divided into five groups of 12 animals each that were distinguished from each other by administration of FK-506 and CTLA4--Ig, that is, isografts interposed in the gap between contralateral posterior tibial nerves (group A, positive control); allografts from C57BL mice implanted without administering any treatment (group B, negative control); allografts from C57BL mice implanted and FK-506 injections administered (group C); allografts from C57BL mice implanted and CTLA4--Ig injections administered (group D); and allografts from C57BL mice implanted and FK-506 and CTLA4--Ig injections administered (group E). Postoperative walking-track functional analysis and histomorphometric studies were then conducted. Data were statistically analysed using the Kruskal-Wallis test. Compared with the negative control (group B), mice treated with both FK-506 and CTLA4--Ig (group E) demonstrated better results at 3 weeks post operation. Similar values were observed in all groups, and there were no statistical differences at 6 weeks post operation. Our results demonstrate that a co-administration of FK-506 and CTLA4--Ig results in functional and histomorphometric recovery that is superior to that seen in the absence of these medications.

Late-onset Increases in Oxidative Stress and Other Tumorigenic Activities and Tumors With a Ha-ras Mutation in the Liver of Adult Male C3H Mice Gestationally Exposed to Arsenic

Tumorigenesis is a complex process involving genetic, epigenetic, and metabolic alterations. Gestational arsenic exposure has been shown to increase hepatic tumors in adult male offspring of C3H mice, which spontaneously develop hepatic tumors often harboring activating Ha-ras mutation. We explored tumor-promoting changes by gestational arsenic exposure with a focus on Ha-ras mutation and gene expression changes. The results of this study demonstrated that gestational arsenic exposure particularly increased hepatic tumors with a C61A Ha-ras mutation. Real-time PCR analyses on the adult normal livers showed that two genes (Creld2, Slc25a30), whose expression are induced by endoplasmic reticulum stress and cellular oxidative stress, respectively, were significantly upregulated and two genes (Fabp4, Ell3), whose products are involved in lipid efflux and apoptosis, respectively, were significantly downregulated more than twofold by gestational arsenic exposure compared with control mice. The expression changes in the four genes were shown to be late-onset events and to some extent to be associated with corresponding histone modifications, and not with DNA methylation changes. The gene expression changes suggested alterations in lipid metabolism and associated oxidative stress augmentation. Consistently, expression of an oxidative-stress-inducible gene heme oxygenase-1 (HO-1) was upregulated in the livers of the arsenic group. We also found increased expression of retrotransposon L1 mRNA in the tumor-bearing livers of the arsenic group in comparison with control mice. These results suggested that gestational arsenic exposure induces tumor-augmenting changes, including oxidative stress and L1 activation, in a late-onset manner, which would particularly promote tumorigenic expansion of cells with a C61A Ha-ras mutation.

Dietary Supplementation with an Extract of North American Ginseng in Adult and Juvenile Mice Increases Natural Killer Cells

Cells belonging to the innate immune system are referred to as natural killer (NK) cells. We recently demonstrated that normal, pre-weaned infant mice, injected with a proprietary extract of ginseng (CVT-E002) had augmented NK cell numbers vs. sham-injected mice. In the present study, we extended these observations into juvenile and adult mice. Thus, young adult (age: 8–9 wk) C3H mice were given daily dietary CVT-E002 for 4 wk followed by untreated chow for the following 2 months, then euthanized (age: 20–21 wk). Other C3H mice (juvenile: 4-wk-old) were given CVT-E002 under the same protocol and sampled at 18 wk of age. In spite of withdrawing the extract 2 months earlier, the absolute numbers of NK cells in the young adults, remained significantly (p < 0.01), and slightly, elevated in the spleen and bone marrow (BM), respectively. The relative numbers (%) of NK cells in the blood also remained elevated (p < 0.05). In juvenile mice fed CVT-E002, the absolute numbers (spleen, BM) and % (blood) of NK cells were all elevated (p<0.01 – p<0.05). The mechanisms responsible for these super-normal numbers of NK cells long after withdrawal of CVT-E002, is as yet unknown.

Absence of intestinal microbiota does not protect mice from diet-induced obesity

The gut microbiota has been implicated in host nutrient absorption and energy homeostasis. We studied the influence of different diets on body composition in germ-free (GF) and conventional (CV) mice. GF and CV male adult C3H mice were fed ad libitum a semi-synthetic low-fat diet (LFD; carbohydrate-protein-fat ratio: 41:42:17; 19.8 kJ/g), a high-fat diet (HFD; 41:16:43; 21.4 kJ/g) or a commercial Western diet (WD; 41:19:41; 21.5 kJ/g). There was no difference in body weight gain between GF and CV mice on the LFD. On the HFD, GF mice gained more body weight and body fat than CV mice, and had lower energy expenditure. GF mice on the WD gained significantly less body fat than GF mice on the HFD. GF mice on both HFD and WD showed increased intestinal mRNA expression of fasting-induced adipose factor/angiopoietin-like protein 4 (Fiaf/Angptl4), but they showed no major changes in circulating Fiaf/Angptl4 compared with CV mice. The faecal microbiota composition of the CV mice differed between diets: the proportion of Firmicutes increased on both HFD and WD at the expense of the Bacteroidetes. This increase in the Firmicutes was mainly due to the proliferation of one family within this phylum: the Erysipelotrichaceae. We conclude that the absence of gut microbiota does not provide a general protection from diet-induced obesity, that intestinal production of Fiaf/Angptl4 does not play a causal role in gut microbiota-mediated effects on fat storage and that diet composition affects gut microbial composition to larger extent than previously thought.

Using Low-Intensity Pulsed Ultrasound to Improve Muscle Healing After Laceration Injury: An in vitro and in vivo Study

The purpose of this study was to determine whether low-intensity pulsed ultrasound (LIPUS) could enhance the regeneration of myofibers and shorten the healing time in injured muscle. NIH C2C12 cells, a well-known myoblastic cell line, are subclones derived from the mouse myoblast cell line established from normal adult C3H mouse leg muscle. The cells differentiate rapidly and produce extensive contracting myotubes expressing characteristic muscle proteins. We exposed C2C12 cells to LIPUS therapy using the EXOGEN 2000+ system ultrasound apparatus (Exogen Inc., Piscataway, NJ, USA) with a total treatment of 20 min every 24 h. At intervals of 2, 4, 6 and 8 days, cell growth was measured by the increase in cell number and western blot analysis of myogenin and actin. Forty mice (C57BL10J+/+) were divided into five groups of eight animals each and used in the published laceration injury model. The gastrocnemius muscle of the left leg was lacerated in all the animals. The control group (sham ultrasound) did not undergo LIPUS therapy. The ultrasound 7-, 14-, 21- and 28-day groups (only changing the number of days during which the ultrasound was applied to the injured muscle) were treated with LIPUS (20 min/day) for 7, 14, 21 and 28 consecutive days, respectively. All animals were sacrificed at 4 weeks after the injury. Evaluation methods included muscle regeneration and muscle contractile properties. LIPUS therapy produced a significantly higher proliferative rate and cell number at days 6 and 8 ( p < 0.05). Densitometric evaluation revealed an increase in myogenin and actin proteins in cells treated with LIPUS in the 4-, 6- and 8-day groups. The regeneration of myofibers, fast-twitch and tetanus of LIPUS-treated muscles (21 and 28 days) was significantly greater relative to control muscles. There was no major strength difference between the normal noninjured muscle and the group treated with LIPUS for 28 days. In conclusion, this was the first experimental study to show that LIPUS therapy is able to enhance the regeneration of myofibers with better physiologic performance in injured mice muscles after laceration, especially prior to postoperative week 4. Findings of this study demonstrate a scientific basis for future clinical trials and establish an indication for LIPUS in enhancing muscle healing after laceration injury. (E-mail: chan512@adm.cgmh.org.tw)

The influence of a chronic adolescent nicotine exposure on ethanol withdrawal severity during adulthood in C3H mice

Animal and human studies have shown tolerance, consumption, relapse, and behavioral interactions between ethanol and nicotine, but little is understood about their interaction, especially as it relates to ethanol withdrawal in adulthood for subjects who have an adolescent history of using these drugs. This study investigated nicotine's influence on ethanol withdrawal seizures in two different age groups of male C3H mice. Adolescent and adult male C3H mice, beginning at postnatal day 28 or 70, respectively, were subjected to a 7-day chronic exposure to ethanol only, ethanol plus nicotine, nicotine only, or vehicle treatment. Six weeks later, all the groups were subjected to chronic exposure to ethanol vapors and the severity of their ethanol withdrawal seizures was assessed by handling-induced convulsions. An adolescent exposure to chronic nicotine resulted in an exacerbation of ethanol withdrawal seizures in adulthood. Given this, adolescence may contain a neurophysiological critical period that is sensitive to nicotine and which may result in an altered response to ethanol dependency in adulthood. These findings have serious implications for the long-term consequences following co-abuse of these drugs during adolescence.

The Effects of Continuous and Intermittent Ethanol Exposure in Adolesence on the Aversive Properties of Ethanol During Adulthood

Alcohol abuse among adolescents is prevalent. Epidemiological studies suggest that alcohol abuse during the adolescent developmental period may result in long-term changes such as an increased susceptibility to alcohol-related problems in adulthood. Laboratory findings suggest that alcohol exposure during the adolescent developmental period, as compared with adulthood, may differentially impact subsequent neurobehavioral responses to alcohol. The present study was designed to examine whether ethanol exposure, continuous versus intermittent, during the adolescent developmental period would alter the aversive properties of ethanol in adult C3H mice. Periadolescent (PD28) male C3H mice were exposed to 64 hours of continuous or intermittent ethanol vapor. As a comparison, adult (PD70) C3H mice were also exposed to 64 hours of continuous or intermittent ethanol vapor. Six weeks after ethanol exposure, taste aversion conditioning was carried out on both ethanol pre-exposed and ethanol-naive animals using a 1-trial, 1-flavor taste-conditioning procedure. Ethanol exposure during the periadolescent period significantly attenuated a subsequent ethanol-induced conditioned taste aversion, as compared with control animals. Adult animals exposed to chronic ethanol vapor during adolescence showed less of an aversion to an ethanol-paired flavor than ethanol-naive adults. Intermittent exposure to ethanol vapor during periadolescence produced a greater attenuation. It is suggested that ethanol exposure during the periadolescent period results in long-term neurobehavioral changes, which lessen a conditioned aversion to ethanol in adulthood. It is suggested that this age-related effect may underlie the increased susceptibility to alcohol-related problems which is negatively correlated with the age of onset for alcohol abuse.

Ketamine exposure in adult mice leads to increased cell death in C3H, DBA2 and FVB inbred mouse strains

Background Drug abuse is common among adolescents and young adults. Although the consequences of intoxication are known, sequelae of drugs emerging on campuses and in clubs nationwide are not. We previously demonstrated that ketamine exposure results in lasting physiological abnormalities in mice. However, the extent to which these deficits reflect neuropathologic changes is not known. Methods The current study examines neuropathologic changes following sub-anesthetic ketamine administration (5 mg/kg i.p. × 5) to three inbred mouse strains. Stereologic quantification of silver stained nuclear and linear profiles as well as activated caspase-3 labeling was used to address: (1) whether or not ketamine increases excitotoxic and apoptotic cell death in hippocampal CA3 and (2) whether or not ketamine-induced cell death varies by genetic background. Results Ketamine increased cell death in hippocampal CA3 of adult C3H, DBA2 and FVB mice. Neither silver staining nor activated caspase-3 labeling varied by strain, nor was there an interaction between ketamine-induced cell death and strain. Conclusions Ketamine exposure among young adults, even in limited amounts, may lead to irreversible changes in both brain function and structure. Loss of CA3 hippocampal cells may underlie persistent ERP changes previously shown in mice and possibly contribute to lasting cognitive deficits among ketamine abusers.

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes

The formation of the neuromuscular junction is characterized by the progressive accumulation of nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane facing the nerve terminal, induced predominantly through the agrin/muscle-specific kinase (MuSK) signaling cascade. However, the cellular mechanisms linking MuSK activation to AChR clustering are still poorly understood. Here, we investigate whether lipid rafts are involved in agrin-elicited AChR clustering in a mouse C2C12 cell line. We observed that in C2C12 myotubes, both AChR clustering and cluster stability were dependent on cholesterol, because depletion by methyl-beta-cyclodextrin inhibited cluster formation or dispersed established clusters. Importantly, AChR clusters resided in ordered membrane domains, a biophysical property of rafts, as probed by Laurdan two-photon fluorescence microscopy. We isolated detergent-resistant membranes (DRMs) by three different biochemical procedures, all of which generate membranes with similar cholesterol/GM1 ganglioside contents, and these were enriched in several postsynaptic components, notably AChR, syntrophin, and raft markers flotillin-2 and caveolin-3. Agrin did not recruit AChRs into DRMs, suggesting that they are present in rafts independently of agrin activation. Consequently, in C2C12 myotubes, agrin likely triggers AChR clustering or maintains clusters through the coalescence of lipid rafts. These data led us to propose a model in which lipid rafts play a pivotal role in the assembly of the postsynaptic membrane at the neuromuscular junction upon agrin signaling.

Periadolescent exposure to ethanol and diazepam alters the aversive properties of ethanol in adult mice

Evidence suggests that the developing adolescent brain may be especially vulnerable to long-term neurobehavioral consequences following ethanol exposure and withdrawal. In the present study, we examined the long-term effect of adolescent ethanol withdrawal on a subsequent EtOH-induced conditioned taste aversion (CTA). Periadolescent and adult C3H mice were exposed to 64 h of continuous (single withdrawal) or intermittent (multiple withdrawal) ethanol vapor. Following each ethanol exposure, animals received either 0, 1, 2, or 3 mg/kg diazepam (DZP) in an attempt to counteract the possible effect of ethanol withdrawal. About 6 weeks following ethanol and DZP treatment, animals were tested for an EtOH-induced CTA. As expected, exposure to EtOH during adolescence attenuated the EtOH-induced CTA as compared to controls. Unexpectedly, administration of DZP during withdrawal did not spare but rather mimicked the attenuation of the EtOH-induced CTA seen in animals exposed to ethanol in adolescence. This attenuation was not evident when EtOH and/or DZP was administered in adulthood. Given the similar mode of action of EtOH and DZP on the GABA system, the principal implication of the present findings is that the intoxicating effect of ethanol on the developing brain can result in long-term changes in the aversive properties of EtOH.

Insulin-like growth factor-1 for amelioration and prevention of radiation-induced kidney dysfunction

9552 Purpose: To test whether insulin-like growth factor-1 (IGF-1) ameliorates or prevents radiation-induced kidney dysfunction and to establish the dose-effect relationship for this growth factor which has not previously been studied in conjunction with kidney irradiation. Background: The kidney is one of the most radiosensitive organs in the abdomen. Therefore, modification of its radiation response might improve the therapeutic ratio in the treatment of retroperitoneal tumors as well as gastric and other malignancies. Material and Methods: Adult female C3H mice were treated with single-fraction radiotherapy to the right kidney with doses of 10–17 Gy ± IGF-1. The kidney function was assessed prior to radiotherapy, 19 weeks thereafter and then every 6 weeks by means of 99mTc-DMSA scans, i.e. static scintigraphy. Maximum follow-up was 12 months. IGF-1 was given subcutaneously either concomitant to radiotherapy or after deterioration of the kidney function, i.e. after 5–6 months. At least 5 mice per group received concomitant IGF-1 starting 24h before irradiation for 3 days or 2 weeks. Delayed treatment after deterioration of the kidney function was administered over 4 weeks, immediately followed by repeat scans every 6 weeks. Doses of IGF-1 were 0.5–25 μg per injection. Results: The function of the irradiated kidney continuously declined during follow-up in all control groups in a dose-dependent fashion. Concomitant treatment with IGF-1 significantly reduced the number of mice with a severe decline, defined as loss of function of 50% or more. In contrast to controls, no statistically significant decline of the mean kidney function was observed in the best IGF-1 group. The best dose of IGF-1 was 5 μg per injection, administered over 2 weeks. Delayed treatment after deterioration of the kidney function was unable to restore the function regardless of the IGF-1 dose. Very few animals with delayed IGF-1 showed at least stabilisation of the compromised function. Conclusion: IGF-1 showed activity as a radioprotective agent when given concomitant to high-dose kidney irradiation. The optimum dose of IGF-1 was 5 μg per injection. In contrast, established renal insufficiency did not improve after IGF-1 treatment. No significant financial relationships to disclose.

Inhibition of the development of metastases by dietary vitamin C:K 3 combination

The tumor growth-inhibiting and chemo-potentiating effects of vitamin C and K 3combinations have been demonstrated both in vitro and in vivo. The purpose of this study was to investigate the influence of orally administered vitamin C and K 3 on the metastasis of mouse liver tumor (T.L.T.) cells implanted in C3H mice. Adult male C3H mice were given water containing vitamin C and K3 (15 g/0.15 g dissolved in 1000 ml) beginning 2 weeks before tumor transplantation until the end of the experiment. T.L.T. cells (106) were implanted intramuscularly in the right thigh of mice. All mice were sacrificed 42 days after tumor transplantation. Primary tumor, lungs, lymph nodes and other organs or tissues suspected of harboring metastases were macroscopically examined. Samples of primary tumors, their local lymph nodes, lungs and main organs such as liver, kidneys, spleen were taken for histological examination. Forty-two percent of control mice exhibited lung metastases and 27% possessed metastases in local lymph nodes whereas 24% of vitamin-treated mice exhibited lung metastases and 10% possessed local lymph nodes metastases. The total number of lung metastases was 19 in control group and 10 in vitamin C and K 3-treated mice. Histopathological examination of the metastatic tumors from the vitamin-treated mice revealed the presence of many tumor cells undergoing autoschizic cell death. These results demonstrate that oral vitamin C and K 3 significantly inhibited the metastases of T.L.T. tumors in C3H mice. At least a portion of this inhibition was due to tumor cell death by autoschizis.

Circadian rhythms in firing rate of individual suprachiasmatic nucleus neurons from adult and middle-aged mice

The suprachiasmatic nucleus contains a biological clock that drives circadian rhythms in vivo and in vitro. It has been suggested that the suprachiasmatic nucleus is a primary target of the aging process, because age-related changes in behavioral rhythms are mirrored in alterations in circadian pacemaker function. Using long-term, single-cell recording, we assessed the effect of age on firing-rate patterns of individual suprachiasmatic nucleus neurons of young adult (2–4 months) and middle-aged (9–11 months) C3H mice. Individual suprachiasmatic nucleus neurons from adult mice maintained in culture for at least one week exhibited robust circadian rhythms in spontaneous activity that were similar in the free-running period (23.7±0.3 h mean±S.E.M.) to recordings from neurons dispersed from neonatal tissue, and showed evidence of entrainment to prior light cycles by exhibiting peak activity, in vitro, approximately 4.0±0.3 h (mean±S.E.M.) after the time of expected light onset. Aging led to a decreased amplitude of impulse activity in dispersed suprachiasmatic nucleus neurons and increased variability in the circadian waveform. From these results we suggest that age-related deterioration in circadian clock function occurs at the level of individual cells, which may account for some of the age-related deficits observed in the expression of behavioral rhythmicity.

Altered Respiratory Activity and Respiratory Regulations in Adult Monoamine Oxidase A-Deficient Mice

The abnormal metabolism of serotonin during the perinatal period alters respiratory network maturation at birth as revealed by comparing the monoamine oxidase A-deficient transgenic (Tg8) with the control (C3H) mice (Bou-Flores et al., 2000). To know whether these alterations occur only transiently or induce persistent respiratory dysfunction during adulthood, we studied the respiratory activity and regulations in adult C3H and Tg8 mice. First, plethysmographic and pneumotachographic analyses of breathing patterns revealed weaker tidal volumes and shorter inspiratory durations in Tg8 than in C3H mice. Second, electrophysiological studies showed that the firing activity of inspiratory medullary neurons and phrenic motoneurons is higher in Tg8 mice and that of the intercostal motoneurons in C3H mice. Third, histological studies indicated abnormally large cell bodies of Tg8 intercostal but not phrenic motoneurons. Finally, respiratory responses to hypoxia and lung inflation are weaker in Tg8 than in C3H mice. dl-p-chlorophenyl-alanine treatments applied to Tg8 mice depress the high serotonin level present during adulthood; the treated mice recover normal respiratory responses to both hypoxia and lung inflation, but their breathing parameters are not significantly affected. Therefore in Tg8 mice the high serotonin level occurring during the perinatal period alters respiratory network maturation and produces a permanent respiratory dysfunction, whereas the high serotonin level present in adults alters the respiratory regulatory processes. In conclusion, the metabolism of serotonin plays a crucial role in the maturation of the respiratory network and in both the respiratory activity and the respiratory regulations.

Topical application of cyclosporin A induces rapid-remodeling of damaged anagen hair follicles produced in cyclophosphamide administered mice

Adult C3H mice which had either anagen IV or anagen VI hair follicles were given the anti-tumor drug cyclophosphamide, and cyclosporin A or minoxidil were topically applied to the mice daily from the 4th day after cyclophosphamide administration. In the mice that had anagen IV-hair follicles, 0.5% cyclosporin A induced very thick and long hairs after 21 days of cyclophosphamide administration, while vehicle and 1% minoxidil induced sparsely visible, short hairs. In the mice which received cyclosporin A, the injured hair follicles seemed to remodel themselves into intact anagen hair follicles and restart the production of hairs, instead of shifting to telogen. In the mice that had anagen VI-hair follicles at the time of cyclophosphamide administration, complete alopecia occurred within the first 7 days in all groups. After 14 days of cyclophosphamide administration, hair regrowth was observed in both the 0.5% cyclosporin A-group and the 1% minoxidil- group with the predominant effect over the vehicle. This study shows that anagen hair follicles respond to cyclophosphamide in different ways depending on their stages (IV and VI), and that the damaged anagen IV hair follicles have the potential of remodeling themselves, which is promoted by topical cyclosporin A administration.

Excitability and contractility of skeletal muscle engineered from primary cultures and cell lines.

The purpose of this study was to compare the excitability and contractility of three-dimensional skeletal muscle constructs, termed myooids, engineered from C2C12 myoblast and 10T1/2 fibroblast cell lines, primary muscle cultures from adult C3H mice, and neonatal and adult Sprague-Dawley rats. Myooids were 12 mm long, with diameters of 0.1-1 mm, were excitable by transverse electrical stimulation, and contracted to produce force. After approximately 30 days in culture, myooid cross-sectional area, rheobase, chronaxie, resting baseline force, twitch force, time to peak tension, one-half relaxation time, and peak isometric force were measured. Specific force was calculated by dividing peak isometric force by cross-sectional area. The specific force generated by the myooids was 2-8% of that generated by skeletal muscles of control adult rodents. Myooids engineered from C2C12-10T1/2 cells exhibited greater rheobase, time to peak tension, and one-half relaxation time than myooids engineered from adult rodent cultures, and myooids from C2C12-10T1/2 and neonatal rat cells had greater resting baseline forces than myooids from adult rodent cultures.