EFFECTS OF PRE- OR POSTNATAL DEXAMETHASONE, ADRENOCORTICOTROPHIC HORMONE AND ENVIRONMENTAL STRESS ON PHENYLETHANOLAMINE N-METHYLTRANSFERASE ACTIVITY AND CATECHOLAMINES IN SYMPATHETIC GANGLIA OF NEONATAL RATS

Abstract— Injections of dexamethasone (0.1 mg/kg/day, s.c.) on the first 2–3 days of life increased the phenylethanolamine- N -methyltransferase (PNMT) activity and epinephrine content of the superior cervical ganglion (SCG) and stellate ganglion of neonatal rats; the dopamine content was unaltered while norepinephrine was slightly reduced in these ganglia. Dexamethasone did not alter the PNMT activity or epinephrine content of the salivary glands or heart. The PNMT activity and epinephrine content of the SCG remained elevated for 10–14 days. Pretreatment with 6-hydroxydopamine did not alter the dexamethasone effects.
Injections of adrenocorticotrophic hormone (ACTH) (25 munits/rat twice a day) or exposure to a cold stress (4°C, 3 times a day) on the first 2–3 days of life, elevated the plasma concentration of corticosterone, and also increased the PNMT activity and epinephrine content in SCG of neonatal rats. Injecting pregnant rats with dexamethasone or ACTH, or exposing them to cold or restraint stress on the last 3 days of gestation increased the PNMT activity and epinephrine content in the SCG of their pups. These results indicate that the actions of dexamethasone on neonatal sympathetic ganglia may be mimicked by increasing the plasma concentration of endogenous adrenocortical steroids.     

A new mathematical model to study bone turnover in growing rats.

A new mathematical model for the study of bone turnover in growing rats was developed. The model predicts a linear relationship between bone mineral content (BMC) and biochemical markers (BMK) of bone turnover assuming that rats are growing, bone turnover is profoundly affected by skeletal maturation, and resorption and formation are physiologically balanced. The model validation was performed by measuring galactosyl-hydroxylysine (GHYL) and hydroxyproline (HYP) in urines. This mathematical evidence supports our proposed use of the specific bone resorption marker GHYL to predict bone mineral content. Further studies on bone turnover will be possible by the application of the same approach.     

Cryopreservation of porcine articular cartilage: MRI and biochemical results after different freezing protocols

The objective of this study was to investigate the effects of cryopreservation on the components of articular cartilage (AC) matrix by utilizing magnetic resonance imaging (MRI) and biochemical assessments. Porcine AC (10mm osteochondral dowels) was collected into four groups - (1) phosphate buffered saline (PBS) control, (2) PBS snap frozen in liquid nitrogen, (3) slow-cooled in dimethyl sulfoxide (DMSO), and (4) slow cooled in PBS (in absence of DMSO). MRI results demonstrated three distinct zones in the cartilage. After exposure to ice formation during cryopreservation procedures, alterations in MRI determined matrix fixed charged density and magnetization transfer rate were noted. In addition, biochemical assays demonstrated significant alterations in chondroitin sulfate and hydroxyproline content over time without differences in hydration or DNA content. In conclusion, MRI was able to detect some changes in the intact cartilage matrix structure consistent with biochemical assessments after ice formation during cryopreservation of intact porcine AC. Furthermore, biochemical assessments supported some of these findings and changed significantly after incubating the cartilage matrix for 36-72 h in PBS in terms of chondroitin sulfate and hydroxyproline content.     

Cyclic estradiol treatment phasically potentiates endogenous cholecystokinin's satiating action in ovariectomized rats.

The influence of ovarian cycling and of exogenous estradiol on the cholecystokinin (CCK) satiety-signalling system was investigated in intact and ovariectomized Long-Evans rats, respectively. Intraperitoneal injection of 1 mg/kg devazepide, the most potent and selective CCK(A) receptor antagonist, increased test meal size during estrus, but not during diestrus, confirming the influence of hypothalamic-pituitary-gonadal function on CCK satiety in intact rats. Devazepide was then tested in ovariectomized rats that received chronic cyclic estradiol (2 microg estradiol benzoate on Tuesday and Wednesday each week) or oil treatment. Devazepide did not increase meal size in estradiol-treated rats on Tuesday, prior to estradiol treatment, compared to oil-treated rats, but did selectively increase meal size on Friday, late in the estradiol replacement cycle, compared to Tuesday, early in the cycle. These results suggest that a phasic potentiation of the endogenous CCK satiety-signalling system is part of the mechanism for the decrease in meal size in female rats during estrus.     

Alizarin red in the study of the apposition and resorption on bone

Human bone 14,000 years of age from the Egyptian Nubian Desert has shown ultrastructural preservation of fossilized fibers with a periodicity of 600 Å. Intact Haversian systems were retained, but hydroxyproline and nitrogen contents were negligible. The fluorine content was markedly elevated. The complex chemical process of fossilization which resulted in retention of gross and micromorphology was examined and indicated the loss of organic material with probable replacement of some hydroxyapatite by amorphous and crystalline carbonates and phosphates.     

Plasma catecholamine responses to tyrosine hydroxylase inhibition and cold exposure

The acute effect of α-methyl-p-tyrosine (αMPT), a tyrosine hydroxylase inhibitor, on plasma levels of norepinephrine (NE), epinephrine (E), dopamine (DA), and adrenal cholesterol content in male rats at room temperature (24°C) and during acute cold exposure (4°C) was evaluated. Compared to saline-treated controls, αMPT: 1) significantly reduced plasma NE and DA in both normal and cold stress conditions, 2) significantly increased plasma E in both environments, and 3) stimulated the adrenal cortex. These findings suggest that tyrosine hydroxylase inhibition and consequent catecholamine synthesis blockade disrupts the homeokinesis of adrenergic processes and may present a significant stress to the intact animal.     

Behavioral and neurochemical investigation of circadian time-place learning in the rat

The ability to form an association between the time and the place of food availability, namely time-place learning, is presumably important for survival. The present study was designed to examine time-place learning and to identify exogenous and endogenous factors that may affect this behavior in rats. In an initial experiment, rats displayed poor time-place behavior and appeared to prefer the feeder that was closer to the center aisle and water supply. When these cues were minimized in a subsequent experiment, rats consistently displayed the time-place discrimination by exhibiting food anticipatory activity (FAA) at the correct location prior to each meal time. These rats also showed significant correlations between the level of FAA and the amount of dopamine turnover (the dihydroxyphenylacetic acid/dopamine ratio) in the nucleus accumbens and paraventricular nucleus of the hypothalamus, indicating possible involvement of regional dopaminergic activity in time-place behavior. No correlation was found for norepinephrine, epinephrine, or serotonin. In addition, the correlation between FAA and dopamine turnover was not found when rats were entrained to only one meal per day. Together, the data suggest that rats can learn the time-place discrimination under proper experimental conditions and that dopamine may play a role in the expression of this behavior.     

Hydrochlorothiazide lowers urinary hydroxyproline in parathyroidectomized rats

This study was undertaken to examine the effects of hydrochlorothiazide treatment on urinary hydroxyproline excretion in parathyroidectomized rats. Urinary hydroxyproline (mumol/24 hr) fell significantly in thiazide-treated rats compared with control animals (5.66 +/- 0.37 versus 7.30 +/- 0.6, P less than 0.05, means +/- SEM). This fall in hydroxyproline excretion occurred without a decrease in glomerular filtration rate. It is concluded that the ability of thiazide diuretics to reduce urinary hydroxyproline excretion is not dependent upon suppression of parathyroid hormone-mediated bone turnover.     

Regulation of hepatic glycogenolysis by glucagon in male and female rats. Role of cAMP and Ca2+ and interactions between epinephrine and glucagon

The effects of 10(-10) to 10(-7) M glucagon on cAMP, phosphorylase a, cell calcium, and glucose production, and glucagon interactions with epinephrine were studied in isolated hepatocytes from adult male and female rats. At physiological concentrations (10(-10) - 10(-9) M), glucagon activated phosphorylase by increasing cAMP and not by raising the cytosolic free calcium. At supra-physiologic concentrations (and in the male only), glucagon slightly increased the cytosolic free calcium, the fractional efflux of calcium, and, after 2 h, decreased the cell calcium content. Exposure of hepatocytes to the simultaneous administration of 10(-9) M glucagon and 10(-7) M epinephrine resulted in a prolongation of the activation of phosphorylase a and a greater release of glucose from glycogen stores than exposure to either agonist alone. In the male, the effects of low concentrations of the two hormones on phosphorylase a activity were additive. Cytosolic free calcium was increased by 10(-6) M epinephrine from 280 to 500 nM while physiological concentrations of glucagon did not change it. In these intact cells, there was no evidence of an alpha 2-adrenergic inhibition of adenyl cyclase and no indication that cAMP depresses the rise in cell calcium induced by alpha-adrenergic stimuli.     

A selective androgen receptor modulator that reduces prostate tumor size and prevents orchidectomy-induced bone loss in rats

The pharmacological activity of JNJ-26146900 is described. JNJ-26146900 is a nonsteroidal androgen receptor (AR) ligand with tissue-selective activity in rats. The compound was evaluated in in vitro and in vivo models of AR activity. It binds to the rat AR with a K(i) of 400nM and acts as a pure androgen antagonist in an in vitro cell-based assay. Its in vitro profile is similar to the androgen antagonist bicalutamide (Casodex). In intact rats, JNJ-26146900 reduces ventral prostate weight with an oral potency (ED(50)) of 20-30mg/kg, again comparable to that of bicalutamide. JNJ-26146900 prevented prostate tumor growth in the Dunning rat model, maximally inhibiting growth at a dose of 10mg/kg. It slowed tumor growth significantly in a CWR22-LD1 mouse xenograft model of human prostate cancer. It was tested in aged male rats for its ability to prevent bone loss and loss of lean body mass following orchidectomy. After 6 weeks of dosing, bone volume decreased by 33% in orchidectomized versus intact vehicle-treated rats with a probability (P) of less than 0.05, as measured by micro-computerized tomography analysis. At a dose of 30mg/kg, JNJ-26146900 significantly reduced castration-induced tibial bone loss as indicated by the following parameters: bone volume, trabecular connectivity, trabecular number and spacing between trabeculae. Bone mineral density decreased from 229+/-34mg/cm(3) of hydroxyapatite to 166+/-26mg/cm(3) following orchidectomy, and was maintained at 194+/-20mg/cm(3) with JNJ-26146900 treatment (P<0.05 relative to orchidectomy alone). Using magnetic resonance imaging, the compound was found to partially prevent orchidectomy-induced loss of lean body mass. Our data show that selective androgen receptor modulators (SARMs) have the potential for anabolic effects on bone and muscle while maintaining therapeutic efficacy in prostate cancer.     

Effects of 900 MHz Radiofrequency Radiation on Skin Hydroxyproline Contents

The present study aimed to investigate the possible effect of pulse-modulated radiofrequency radiation (RFR) on rat skin hydroxyproline content, since skin is the first target of external electromagnetic fields. Skin hydroxyproline content was measured using liquid chromatography mass spectrometer method. Two months old male wistar rats were exposed to a 900 MHz pulse-modulated RFR at an average whole body specific absorption rate (SAR) of 1.35 W/kg for 20 min/day for 3 weeks. The radiofrequency (RF) signals were pulse modulated by rectangular pulses with a repetition frequency of 217 Hz and a duty cycle of 1:8 (pulse width 0.576 ms). A skin biopsy was taken at the upper part of the abdominal costa after the exposure. The data indicated that whole body exposure to a pulse-modulated RF radiation that is similar to that emitted by the global system for mobile communications (GSM) mobile phones caused a statistically significant increase in the skin hydroxyproline level (p = 0.049, Mann–Whitney U test). Under our experimental conditions, at a SAR less than the International Commission on Non-Ionizing Radiation Protection safety limit recommendation, there was evidence that GSM signals could alter hydroxyproline concentration in the rat skin.     

The effect of chronic nicotine administration on bone mineral content and bone strength in normal and castrated male rats.

Tobacco, containing nicotine as the principal pharmacologically active chemical, has been identified as being a risk factor for the development of osteoporosis. Thirty-two male Wistar rats of two months of age were castrated or sham operated to evaluate the effects of long-term administration (four months) of nicotine in drinking water (9.0 mg/kg/day). The presence of cotinine in urine confirmed successful delivery of nicotine. The bones were tested mechanically by a three point bending test in a Mini Bionix (MTA) testing system. The bones from castrated rats were characterized by a reduction in bone density as well as ash, calcium and phosphate content. Castration significantly altered mechanical properties of bone (9%) and femoral cortical thickness. When intact rats were treated with a high dose of nicotine, nicotine had negative effect on tibial bone density as well as ash, calcium, phosphate content and significantly altered the mechanical properties of bone (12%) and femoral cortical thickness compared to intact animals. Nicotine itself does not exert any anti-androgenic effect and does not produce changes in the weight of seminal vesicles. Nicotine-induced bone loss is associated with high bone turnover in the male rats as expressed by increased TrACP and B-ALP. When castrated rats were treated with the high dose of nicotine the changes in bone density resulting from castration were not further potentiated. These results document the efficacy of nicotine at high doses to cause bone loss and loss of bone mechanical strength in intact rats. The results of the present study may be interpreted as supporting the hypothesis of nicotine as a risk factor for osteoporosis.     

Silicon deprivation decreases collagen formation in wounds and bone,and ornithine transaminase enzyme activity in liver

We have shown that silicon (Si) deprivation decreases the collagen concentration in bone of 9-wk-old rats. Finding that Si deprivation also affects collagen at different stages in bone development, collagen-forming enzymes, or collagen deposition in other tissues would have implications that Si is important for both wound healing and bone formation. Therefore, 42 rats in experiment 1 and 24 rats in experiment 2 were fed a basal diet containing 2 or 2.6 μg Si/g, respectively, based on ground corn and casein, and supplemented with either 0 or 10 μg Si/g as sodium metasilicate. At 3 wk, the femur was removed from 18 of the 42 rats in experiment 1 for hydroxyproline analysis. A polyvinyl sponge was implanted beneath the skin of the upper back of each of the 24 remaining rats. Sixteen hours before termination and 2 wk after the sponge had been implanted, each rat was given an oral dose of ¹⁴C-proline (1.8 μCi/100 g body wt). The total amount of hydroxyproline was significantly lower in the tibia and sponges taken from Si-deficient animals than Si-supplemented rats. The disintegrations per minute of ¹⁴C-proline were significantly higher in sponge extracts from Si-deficient rats than Si-supplemented rats. Additional evidence of aberrations in proline metabolism with Si deprivation was that liver ornithine aminotransferase was significantly decreased in Si-deprived animals in experiment 2. Findings of an increased accumulation of ¹⁴C-proline and decreased total hydroxyproline in implanted sponges and decreased activity of a key enzyme in proline synthesis (liver ornithine aminotransferase) in Si-deprived animals indicates an aberration in the formation of collagen from proline in sites other than bone that is corrected by Si. This suggests that Si is a nutrient of concern in wound healing as well as bone formation. The U.S. Department of Agriculture, Agricultural Research Service, Northern Plains Area is an equal opportunity/affirmative action employer, and all agency services are available without discrimination. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.     

Lead levels in bone and hair of rats treated with lead acetate

The use of hair and bone as media in evaluation of lead exposure was investigated in this study. For 12–16 wk rats were given tap water containing lead acetate in the following concentrations: 41.7 mg Pb/L, 83.3 mg Pb/L, and 166.6 mg Pb/L. The animals were sacrificed every 4 wk and their tibia bones and hair were collected for determination of lead content. In control animals, the lead level amounted to 1.2 μg/g (range 0.8–1.3 μg/g) and 0.7 μg/g (range 0.4–2.0 μg/g) in bone and hair, respectively. In the treated rats the accumulation of lead in bone and hair occurred in a dose-dependent manner. A positive corelation (r=0.876) was established between the lead levels in bone and hair of the rats. The regression equation was as follows: μg Pb/g bone=0.842×μg Pb/g hair+1.868. After discontinuation of exposure, a significant decrease in the lead content in bone and hair was noticed. About 9 wk after cessation of treatment, the lead content in hair declined to the pre-exposure level, but 64% of the maximal lead concentration did remain in bone. The results of this study indicate that during a continuous exposure the lead level in hair reflects its content in bone. Such phenomena did not occur during the postexposure period.     

Inhibition of tissue repair by spironolactone: Role of mineralocorticoids in fibrous tissue formation

Mineralocorticoids have been implicated in promoting fibrous tissue formation in various organs. In the present study, we sought to address the potential contribution of mineralocorticoids to fibrous tissue formation using a skin pouch model which has proved valuable for the analysis of inflammatory and wound healing responses. Skin pouches were induced in rats by administration of a phorbol ester, croton oil (0.5 ml of a 1% solution). After 2 weeks, rats were killed and intact pouch tissue collected. Pouch weights of control and aldosterone-treated (0.75 g/h via osmotic minipump) rats were similar (3.33 ± 0.44 g vs. 3.70 ± 0.28 g respectively). However, pouch weights were reduced by more than 50% in spironolactone-treated (25 mg/day powdered in food) animals (1.62 ± 0.22 g and 1.27 ± 0.23 g respectively in aldosterone and spironolactone alone groups). To ascertain the effects of different treatments on collagen accumulation, hydroxyproline concentration was measured. Compared with controls, hydroxyproline concentration was significantly reduced following spironolactone treatment (17.1 ± 0.08 vs. 7.5 ± 2.0 g/mg dry wt, respectively, p < 0.01). This response to spironolactone was negated by coadministration of aldosterone (hydroxyproline concentration was 18.6 ± 2.1 g/mg dry wt). Following bilateral adrenalectomy, spironolactone reduced pouch weight and hydroxyproline concentration, which was not the case for adrenalectomy alone. Two week aldosterone administration in uninephrectomized rats on high salt diet was deemed ineffective in modulating pouch development (pouch wet wts were 3.48 ± 0.4 g vs. 3.00 ± 0.19 g in controls and aldosterone-treated rats, respectively). Mineralocorticoid receptor expression in pouch tissue was demonstrated by RT/PCR. Furthermore, NADP+-dependent 11-hydroxysteroid dehydrogenase 1 (11-HSD1) activity was detected in pouch tissue, together with lower levels of NAD+-dependent 11-HSD2. Spironolactone (p < 0.05) significantly reduced 11-HSD1 activity compared with controls. Thus, fibrous tissue possesses requisite components of MC action, and antagonism of mineralocorticoid receptors by spironolactone attenuates its formation. Pouch formation is under the influence of circulating MC and, we would like to propose, is also mediated through corticosteroids generated de novo at the site of tissue repair.     

A second moment NMR method to study phase changes in bone

A second moment NMR method is presented to study changes involving phosphorus in intact bone. On the assumption that the inorganic calcium phosphate system in bone is a mixture of hydroxyapatite and tricalcium phosphate, the percentage of each is determined for cow bone and two human samples. Comparing this method with an X-ray radial distribution function method gives promising results.     

Effect of exercise on the development of osteoporosis in adult rats

The role of moderate exercise in the prevention of high-turnover osteoporosis was investigated by the use of an animal model. The effect of chronic training on gravimetric, mineral, physical, and histological parameters of normal bone was also examined. Fifty-six adult female Long-Evans rats were divided into four groups: sedentary (C) and exercising controls (E) and sedentary (O) and exercising osteoporotics (EO). Exercising animals ran 4 h/wk for 1 yr. Two percent NH4Cl added to drinking water induced osteoporosis as shown by significantly lower femoral density and breaking strength and histomorphometrically quantified tibial trabecular bone volume but a normal mineral-to-matrix ratio in the O rats. The development of high-turnover osteoporosis in O rats was confirmed by significantly higher alkaline phosphatase activity (P less than 0.05), urinary hydroxyproline content (P less than 0.01), resorption surfaces (P less than 0.01), and histological parameters of bone formation (P less than 0.01). Exercise prevented all these biochemical, biophysical, and histological abnormalities in the EO group. Exercise had no influence on the density of normal femurs but tended to increase their breaking strength (by 11%) compared with femurs of C rats (P = 0.11).     

Effects of 0.4 T rotating magnetic field exposure on density, strength, calcium and metabolism of rat thigh bones

The current study investigated the effects of 0.4 T rotary non-uniform magnetic field (RMF) exposure on bone density in ovariectomized (OVX) rats. Results showed that many bone indexes are significantly elevated after RMF exposure compared to the control OVX group and confirmed mechanistic evidence that strong magnetic field (MF) exposure could effectively increase bone density and might be used to treat osteoporosis. Synergy of daily RMF exposure (30 min a day for 30 days using an 8 Hz rotary 0.4 T MF) with calcium supplement tended to increase the indexes of thigh bone density, energy absorption, maximum load, maximum flexibility, and elastic deformation as compared to those of untreated OVX control group. Results also revealed that the indexes of alkaline phosphatase (ALP), serum phosphate, and serum calcium were higher in rats exposed to RMF with calcium than in the untreated OVX control group. Changes in bone mineral density (BMD) and bone mineral content (BMC) were observed in rats for three months including the first month RMF exposure. Bone density in rats exposed each day for 60 min increased during 1-month exposure and continued to increase during the post-exposure period. Furthermore, bone density and calcium content in rats exposed for 90 min daily decreased initially in the exposure month; however, ratio of increase was well above the control values by the end of the post-exposure period suggesting possible window and delayed effects. The study indicated that RMF exposure to both male and OVX female rats for 120 min a day over 15 day period should effectively promote increase of bone calcium contents (BCC) and bone-specific alkaline phosphatase (BAP) in rats thigh bone as well as a corresponding decrease in deoxypyridinoline crosslinks (DPD).     

Role of the sympathetic nervous system in cold-induced hypertension in rats

Hypertension develops in rats exposed chronically to cold [6 +/- 2 degrees C (SE)] and includes both an elevation of mean arterial pressure and cardiac hypertrophy. Previous studies suggest that cold-exposed animals, at least initially, have a large sustained increase in the activity of their sympathetic nervous system, suggesting a failure of the baroreceptor system to provide sufficient negative feedback to the central nervous system. The present study was designed to investigate whether alterations in the activity of the sympathetic nervous system, including the baroreceptor reflex, occur during exposure to cold and whether they contribute to cold-induced hypertension. Twenty male rats were prepared with indwelling catheters in the femoral artery and vein. Ten of the rats were exposed to cold (6 +/- 2 degrees C) chronically, while the remaining 10 were kept at 26 +/- 2 degrees C. Withdrawal of arterial blood samples (less than 5 ml/kg), measurement of direct arterial pressures, and measurement of baroreflex function were carried out at 0800 h at intervals throughout the experiment. Norepinephrine and epinephrine concentrations in plasma were also determined at intervals throughout the experiment. Systolic, diastolic, and mean blood pressures of cold-exposed rats were increased to levels significantly above those of controls. The sensitivity of the baroreflex (delta heart period/delta mean arterial pressure) was decreased in the cold-treated group. The concentration of norepinephrine in plasma increased after 24 h of exposure to cold and remained elevated throughout the experiment, whereas the concentration of epinephrine in plasma increased initially but returned to control levels after 19 days of exposure to cold.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antifibrogenic effect in vivo of low doses of insulin-like growth factor-I in cirrhotic rats.

Insulin-like growth factor-I (IGF-I) is produced mainly in the liver and it induces beneficial effects on the nutritional status, the liver function and oxidative hepatic damage in cirrhotic rats. The aim of this work was to analyze the effect of IGF-I on mechanisms of fibrogenesis in cirrhotic rats. Liver cirrhosis was induced by CCl(4) inhalation and phenobarbital in Wistar rats. Ten days after stopping CCl(4) administration (day 0), rats received either IGF-I (2 microg/100 g bw/day) (CI+IGF) or saline (CI) subcutaneously during 14 days. Animals were sacrificed on day 15. As control groups were used: healthy rats (CO) and healthy rats treated with IGF-I (CO+IGF). Liver histopathology, hydroxyproline content, prolyl hydroxylase activity, collagen I and III mRNA expression and the evolution of transformed Ito cells into myofibroblasts were assessed. Among the two control groups (CO+IGF), no differences were found in hydroxyproline content and these levels were lower than those found in the two cirrhotic groups. Compared with untreated cirrhotic rats, the CI+IGF-I animals showed a significant reduction in hydroxyproline content, prolyl hydroxylase activity and collagen alpha 1(I) and alpha1(III) mRNA expression. A higher number of transformed Ito cells (alpha-actin +) was observed in untreated cirrhotic animals as compared to CO and CI+IGF groups. In summary, treatment with IGF-I reduced all of the studied parameters of fibrogenesis. In conclusion, low doses of IGF-I induce in vivo an antifibrogenic effect in cirrhotic rats.