Infrared Laser-Mediated Gene Induction at the Single-Cell Level in the Regenerating Tail of Xenopus laevis Tadpoles.

We describe a precise and reproducible gene-induction method in the amphibian, Xenopus laevis Tetrapod amphibians are excellent models for studying the mechanisms of three-dimensional organ regeneration because they have an exceptionally high regenerative ability. However, spatial and temporal manipulation of gene expression has been difficult in amphibians, hindering studies on the molecular mechanisms of organ regeneration. Recently, however, development of a Xenopus transgenic system with a heat-shock-inducible gene has enabled the manipulation of specific genes. Here, we applied an infrared laser-evoked gene operator (IR-LEGO) system to the regenerating tail of Xenopus tadpoles. In this method, a local heat shock by laser irradiation induces gene expression at the single-cell level. After amputation, Xenopus tadpoles regenerate a functional tail, including spinal cord. The regenerating tail is flat and transparent enabling the targeting of individual cells by laser irradiation. In this protocol, a single neural progenitor cell in the spinal cord of the regenerating tail is labeled with heat-shock-inducible green fluorescent protein (GFP). Gene induction at the single-cell level provides a method for rigorous cell-lineage tracing and for analyzing gene function in both cell-autonomous and noncell-autonomous contexts. The method can be modified to study the regeneration of limbs or organs in other amphibians, including Xenopus tropicalis, newts, and salamanders.

Effects of chronic exposure to radiofrequency electromagnetic fields on energy balance in developing rats.

The effects of radiofrequency electromagnetic fields (RF-EMF) on the control of body energy balance in developing organisms have not been studied, despite the involvement of energy status in vital physiological functions. We examined the effects of chronic RF-EMF exposure (900 MHz, 1 V m(-1)) on the main functions involved in body energy homeostasis (feeding behaviour, sleep and thermoregulatory processes). Thirteen juvenile male Wistar rats were exposed to continuous RF-EMF for 5 weeks at 24 °C of air temperature (T a) and compared with 11 non-exposed animals. Hence, at the beginning of the 6th week of exposure, the functions were recorded at T a of 24 °C and then at 31 °C. We showed that the frequency of rapid eye movement sleep episodes was greater in the RF-EMF-exposed group, independently of T a (+42.1 % at 24 °C and +31.6 % at 31 °C). The other effects of RF-EMF exposure on several sleep parameters were dependent on T a. At 31 °C, RF-EMF-exposed animals had a significantly lower subcutaneous tail temperature (-1.21 °C) than controls at all sleep stages; this suggested peripheral vasoconstriction, which was confirmed in an experiment with the vasodilatator prazosin. Exposure to RF-EMF also increased daytime food intake (+0.22 g h(-1)). Most of the observed effects of RF-EMF exposure were dependent on T a. Exposure to RF-EMF appears to modify the functioning of vasomotor tone by acting peripherally through α-adrenoceptors. The elicited vasoconstriction may restrict body cooling, whereas energy intake increases. Our results show that RF-EMF exposure can induce energy-saving processes without strongly disturbing the overall sleep pattern.

Effects of X-ray irradiation of female mice in preconceptive period on polymorphism of simple repeats in DNA of offspring of different sex.

Sibs groups of F1-offspring born by non-irradiated mice and by female mice exposed to X-ray radiation in preconceptive period (50-200 cGy) were compared. Arbitrary primed PCR revealed significantly increased polymorphism of simple DNA repeats in somatic tissues of the offspring from female mice irradiated in a dose of 200 cGy. The increase in DNA polymorphism in postmitotic brain tissues and in peripheral blood was more pronounced than in proliferating spleen tissues and in the epithelium of tail tip. In the tissues of female offspring from irradiated mothers, higher increase in DNA polymorphism was observed in comparison with the tissues of male offspring from the same mothers.

Focused RF hyperthermia using magnetic fluids.

Heat therapies such as hyperthermia and thermoablation are very promising approaches in the treatment of cancer. Compared with available hyperthermia modalities, magnetic fluid hyperthermia (MFH) yields better results in uniform heating of the deeply situated tumors. In this approach, fluid consisting of superparamagnetic particles (magnetic fluid) is delivered to the tumor. An alternating (ac) magnetic field is then used to heat the particles and the corresponding tumor, thereby ablating it. However, one of the most serious shortcomings of this technique is the unwanted heating of the healthy tissues. This results from the magnetic fluid diffusion from the tumor to the surrounding tissues or from incorrect localization of the fluids in the target tumor area. In this study, the authors demonstrated that by depositing appropriate static (dc) magnetic field gradients on the alternating (ac) magnetic fields, focused heating of the magnetic particles can be achieved. A focused hyperthermia system was implemented by using two types of coils: dc and ac coils. The ac coil generated the alternating magnetic field responsible for the heating of the magnetic particles; the dc coil was used to superimpose a static magnetic field gradient on the alternating magnetic field. In this way, focused heating of the particles was obtained in the regions where the static field was dominated by the alternating magnetic field. In vitro experiments showed that as the magnitude of the dc solenoid currents was increased from 0 to 1.8 A, the specific absorption rate (SAR) of the superparamagnetic particles 2 cm apart from the ac solenoid center decreased by a factor of 4.5, while the SAR of the particles at the center was unchanged. This demonstrates that the hyperthermia system is capable of precisely focusing the heat at the center. Additionally, with this approach, shifting of the heat focus can be achieved by applying different amounts of currents to individual dc solenoids. In vivo experiments were performed with adult rats, where magnetic fluids were injected percutaneously into the tails (with homogeneous fluid distribution inside the tails). Histological examination showed that, as we increased the dc solenoid current from 0.5 to 1.8 A, the total burned volume decreased from 1.6 to 0.2 cm3 verifying the focusing capability of the system. The authors believe that the studies conducted in this work show that MFH can be a much more effective method with better heat localization and focusing abilities.

Application of a temperature-dependent fluorescent dye (Rhodamine B) to the measurement of radiofrequency radiation-induced temperature changes in biological samples.

We have applied a non-contact method for studying the temperature changes produced by radiofrequency (RF) radiation specifically to small biological samples. A temperature-dependent fluorescent dye, Rhodamine B, as imaged by laser scanning confocal microscopy (LSCM) was used to do this. The results were calibrated against real-time temperature measurements from fiber optic probes, with a calibration factor of 3.4% intensity change degrees C(-1) and a reproducibility of +/-6%. This non-contact method provided two-dimensional and three-dimensional images of temperature change and distributions in biological samples, at a spatial resolution of a few micrometers and with an estimated absolute precision of around 1.5 degrees C, with a differential precision of 0.4 degree C. Temperature rise within tissue was found to be non-uniform. Estimates of specific absorption rate (SAR) from absorbed power measurements were greater than those estimated from rate of temperature rise, measured at 1 min intervals, probably because this interval is too long to permit accurate estimation of initial temperature rise following start of RF exposure. Future experiments will aim to explore this.

Cerebral columnar organization of the first nociceptive component induced by CO2 laser on the tail of the rat.

The somatotopic map of the first nociceptive component in the primary somatosensory cortex (S1) is still unclear. In this study, a CO(2) laser was applied to the tail of the rat to induce nociception without the interference from large myelinated (A(beta)) fibers. Thus, only noxious fibers could be activated. Two-dimensional current-source-density analysis was used to analyze the evoked field potentials. Using this method, the nociceptive responses of A(delta)-fibers in S1 were verified, and the somatotopic map of the first nociceptive component in S1 was identified. We found that whether light touch or laser-induced nociception was applied to the tail of the rat, the responsive topography in S1 was consistent. Discrimination of these two modalities was achieved vertically in the same column; the deeper layer represented the nociceptive response while the superficial layer encoded the response to light touch. This is quite different from that of a primate brain.

Effect of chronic intermittent exposure to AM radiofrequency field on responses to various types of noxious stimuli in growing rats.

There are several reports of altered pain sensation after exposure (from a few minutes to hours in single or repeated doses for 2-3 weeks) to electromagnetic fields (EMF) in adults. The commonly utilized noxious stimulus is radiant heat. The nociceptive responses are known to be influenced by characteristics of stimulus, organism, and environment. We studied the pattern of nociceptive responses to various noxious stimuli in growing rats exposed to radiofrequency field (73.5 MHz amplitude modulated, 16 Hz power density 1.33 mw/cm(2), SAR = 0.4 w/kg) for 45 d (2 h/d). Threshold current for stimulation of nociceptive afferents to mediate motor response of tail (TF), vocalization during stimulus (VD), and vocalization after discharge (VA); the withdrawal latency of tail (TFL) and hind paw (HPL) to thermal noxious stimulus and tonic pain responses were recorded in every rat. The TFL was not affected, HPL was decreased (p < 0.01), and the thresholds of TF and VD were not affected, while, that of VA was significantly decreased. The tonic pain rating was decreased (p < 0.01). A decrease in the threshold of VA (p < 0.01) is indicative of an increase in the emotional component of the response to the phasic pain, whereas a decrease in the pain rating indicates analgesia in response to the tonic pain. The results of our study suggest that chronic (45 d), intermittent (2 h/d) amplitude modulated RF field exposure to the peripubertal rat increases the emotional component of phasic pain over a basal eaualgesic state, while late response to tonic pain is decreased. The data suggest that amplitude modulated RF field differentially affects the mechanisms involved in the processing of various noxious stimuli.

Pre-emptive ring-block with bupivacaine prevents the development of thermal hyperalgesia, but not sustained mechanical hyperalgesia, in rat tails exposed to ultraviolet A light.

UNLABELLED: We investigated the role of the C-fiber barrage in the development of hyperalgesia in rat tails exposed to ultraviolet A (UVA)-light exposure by pre-emptively blocking C-fiber activation with the local anesthetic bupivacaine. Thirty minutes before UVA-light exposure, male Sprague-Dawley rats were given subcutaneous injections, in the base of the tail, of either saline or bupivacaine (1 mL of .5%). Thermal hyperalgesia was assessed daily from day 1 to day 10 after UVA-light exposure by measuring response latency to noxious heat (tail immersion in 49 degrees C water). Injection of bupivacaine completely prevented the development of thermal hyperalgesia (P < .05, main effect of group, 2-way analysis of variance). Primary mechanical hyperalgesia was assessed daily from day 1 to day 14 after UVA-light exposure by measuring aversive behavior responses to a punctate pressure applied to the tail with a von Frey anesthesiometer. The rats given bupivacaine developed hyperalgesia to the mechanical challenge that persisted for 14 days (P < .05, main effect of time, 2-way analysis of variance) and was identical to the hyperalgesia developed by the rats given saline. We concluded that the C-fiber barrage is involved in the development of thermal hyperalgesia but not sustained primary mechanical hyperalgesia, induced by exposing rats' tails to UVA light. PERSPECTIVE: UVA-light exposure of the rat tail causes prolonged hyperalgesia to noxious thermal and mechanical challenges. We have demonstrated that the C-fiber barrage is involved in the development of sustained thermal hyperalgesia, but not mechanical hyperalgesia, caused by exposure of the rat tail to UVA light.

Behavioral control of the stressor modulates stress-induced changes in neurogenesis and fibroblast growth factor-2.

The controllability of stressors modulates many of the consequences of stressor exposure. Here, we used immunohistochemistry to examine neural progenitor cell proliferation and survival and basic fibroblast growth factor-2 in the hippocampus of male rats after controllable or uncontrollable tailshock. A series of identical tailshocks were delivered to yoked pairs of rats. One rat could terminate shocks to both rats of the pair. Reductions in neural progenitor cells were observed at 1-2 days and at 28 days in rats exposed to uncontrollable shock. Controllable shock produced an increase in fibroblast growth factor-2 in the dentate gyrus and CA1 2 h after stress and in the dentate gyrus 24 h after stress. Thus, stressor controllability modulates stress-induced decreases in neurogenesis and increases in fibroblast growth factor-2.

Exposure of the rat tail to ultraviolet A light produces sustained hyperalgesia to noxious thermal and mechanical challenges.

We aimed to establish whether exposing the tails of rats to ultraviolet A (UVA) light generated sustained hyperalgesia to noxious thermal and mechanical challenges. The tails of 21 rats underwent eight 40s exposures of UVA light, with 260s between each exposure. As a control procedure, during UVA-light exposure the tails of 11 of those rats were shielded with aluminium foil. Thermal hyperalgesia was assessed by immersing the rat tail in 49 degrees C water (modified tail flick test). Mechanical hyperalgesia was assessed by applying a bar algometer onto the tail and timing the escape response. Exposure to direct UVA light produced hyperalgesia for 8 days to the noxious thermal challenge (P<0.05, two-way ANOVA, Tukey post hoc tests) and at least 16 days to the noxious mechanical challenge (P<0.05, two-way ANOVA, Tukey post hoc tests). They gained mass throughout the study at the same rate as the control rats. The control rats did not develop thermal nor mechanical hyperalgesia. The tails of a further 20 rats were exposed similarly, and tail tissue examined histologically. Both exposed and control rats developed mild chronic inflammation unrelated to the hyperalgesia.

Effect of number of tailshocks on learned helplessness and activation of serotonergic and noradrenergic neurons in the rat.

Adult male albino rats were exposed to varying numbers of tailshocks (0, 10, 50 or 100). The following day, their escape latencies in a shuttlebox were measured in order to estimate the degree of learned helplessness (LH) produced by the varying number of shocks. Only the groups exposed to 50 or 100 shocks displayed evidence of LH. In a parallel experiment, c-fos activation was used to determine the degree of activation of raphe serotonergic neurons (FosIR+5-HT) and locus coeruleus (LC) noradrenergic neurons (FosIR+TH) produced by the same shock conditions. Compared to unhandled cage controls, all shock groups (0 shocks was a restrained group) significantly activated both raphe and LC neurons. The 50 and 100 shock groups had significantly higher degrees of activation of serotonergic neurons in the rostral raphe groups and the LC than the 0 and 10 shock groups. These data are consistent with the hypothesis that activation of rostral raphe serotonergic neurons and LC noradrenergic neurons beyond a certain threshold may be critical for the development of LH. The relevance of these results for elucidating the neural bases of psychopathology is discussed.

Effects of carbon-ion radiation on the postnatal development of mice and on the yield of white spots in the mid-ventrum and tail tips.

Pregnant female C57BL/10JHir mice were irradiated whole-body at 9 days of gestation with a single acute dose of carbon-ion radiation. The average linear energy transfer (LET) of the carbon ions was 50 keV/microm within a spread-out Bragg peak (SOBP). The effects were studied by scoring changes in the postnatal development of the mice as well as in the pigmentation of the cutaneous coats and tail tips of their offspring 22 days after birth. The percentage of live births was reduced in mice exposed to carbon ions at doses greater than 0.5 Gy. The survival to day 22 was also reduced in mice exposed to carbon ions at doses greater than 0.75 Gy. Moreover, the body weight at day 22 was reduced in mice exposed to carbon ions at doses greater than 0.1 Gy. A comparison of the survival to day 22 after exposure to carbon ions with our previous results for 60Co gamma rays indicated that carbon ions were twice as effective as gamma rays. White spots were found in the mid-ventrum as well as in the tail tips of offspring exposed to carbon ions in utero. The frequency and the size of the white spots in the mid-ventrum and in the tail tips increased as the dose increased. Carbon ions appear to be slightly more effective than the gamma rays used in our previous study. In the ventral white spots, no melanocytes were observed in the epidermis, dermis and hair follicles. These results indicate that prenatal exposure to carbon ions has a greater effect on the postnatal development and survival of mice than does exposure to gamma rays, and that the relative biological effectiveness is greater than that for effects on melanocyte development.

Identification of radiation-sensitive mutants in the Medaka, Oryzias latipes.

We screened populations of N-ethyl-N-nitrosourea (ENU)-mutagenized Medaka, (Oryzias latipes) for radiation-sensitive mutants to investigate the mechanism of genome stability induced by ionizing radiation in developing embryos. F3 embryos derived from male founders that were homozygous for induced the mutations were irradiated with gamma-rays at the organogenesis stage (48hpf) at a dose that did not cause malformation in wild-type embryos. We screened 2130 F2 pairs and identified three types of mutants with high incidence of radiation-induced curly tailed (ric) malformations using a low dose of irradiation. The homozygous strain from one of these mutants, ric1, which is highly fertile and easy to breed, was established and characterized related to gamma-irradiation response. The ric1 strain also showed higher incidence of malformation and lower hatchability compared to the wild-type CAB strain after gamma-irradiation at the morula and pre-early gastrula stages. We found that the decrease in hatching success after gamma-irradiation, depends on the maternal genotype at the ric1 locus. Terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end-labeling assays showed a high frequency of apoptosis in the ric1 embryos immediately after gamma-irradiation at the pre-early gastrula stage but apoptotic cells were not observed before midblastula transition (MBT). The neutral comet assay revealed that the ric1 mutant has a defect in the rapid repair of DNA double-strand breaks induced by gamma-rays. These results suggest that RIC1 is involved in the DNA double strand break repair in embryos from morula to organogenesis stages, and unrepaired DNA double strand breaks in ric1 trigger apoptosis after MBT. These results support the use of the ric1 strain for investigating various biological consequences of DNA double strand breaks in vivo and for sensitive monitoring of genotoxicity related to low dose radiation.

Identification of a novel frog RFamide and its effect on the latency of the tail-flick response of the newt.

Neuropeptide FF, one of the mammalian PQRFamides, has been reported to affect the latency of the tail-flick response in rat. We intended to examine the nociceptive effect by the peptide PQRFamides from the comparative aspect. Using the dot immunoblot method with antiserum to FMRFamide as an assay system, a peptide (frog's nociception-related peptide, fNRP) which has the C-terminal sequence PQRFamide was isolated from the brain of the frog, Rana catesbeiana. The determined sequence, SIPNLPQRF-NH(2), is the same as that named first (frog growth hormone-releasing peptide-gene-related peptide-1: fGRP-RP-1, which is encoded in the cDNA of the fGRP precursor. Since the peptide was isolated from the frog brain, we tested another amphibian, the newt, which has a tail, by the hot beam tail-flick test. Intraperitoneal injection of fNRP significantly increased the latency of the pain response (tail-flick) 90 min after administration. The effect was blocked by simultaneous administration of 5 mM naloxone. The result provides evidence for the interaction of fNRP and opioid steps in the analgesia pathways in the newt.

In vivo response to the different LET regions of a 55 MeV helium ion beam.

We evaluated the in vivo response to heavy particle irradiation in rat tail epidermis using silver-stained nucleolar organizer regions (AgNOR) as the end-point. The energy degradation of the beam across the circular section of the tail allowed us to study the damage elicited by two different LET regions of a helium beam, i.e. non-Bragg peak (NBP) and Bragg peak (BP), at different sites on the same sample. The tails were locally irradiated with a helium ion beam at different fluences. AgNOR exhibited marked variations between tissue areas only a few micrometers apart within each tail exposed to a given beam fluence. An analysis of the AgNOR variations in NBP and BP areas of tails exposed to different beam fluences revealed a dose-dependent effect. The AgNOR provide quantitative evidence of differential damage in neighboring tissue areas exposed to different LET regions of a helium-ion beam.

Cardiovascular and thermal effects of microwave irradiation at 1 and/or 10 GHz in anesthetized rats.

Relatively large thermal gradients may exist during exposure of an animal to microwaves (MWs), particularly at high frequencies. Differences in thermal gradients within the body may lead to noticeable differences in the magnitude of cardiovascular changes resulting from MW exposure. This study compares the thermal distribution and cardiovascular effects of exposure to a single MW frequency with effects of simultaneous exposure to two frequencies. Ketamine-anesthetized male Sprague-Dawley rats (n = 58) were exposed individually to one of three conditions: 1-GHz, 10-GHz, or combined 1- and 10-GHz MWs at an equivalent whole-body specific absorption rate of 12 W/kg. The continuous-wave irradiation was conducted under far-field conditions with animals in E orientation (left lateral exposure, long axis parallel to the electric field) or in H orientation (left lateral exposure, long axis perpendicular to the electric field). Irradiation was started when colonic temperature was 37.5 degrees C and was continued until lethal temperatures were attained. Colonic, tympanic, left and right subcutaneous, and tail temperatures, and arterial blood pressure, heart rate, and respiratory rate were continuously recorded. In both E and H orientations, survival time (i.e., time from colonic temperature of 37.5 degrees C until death) was lowest in animals exposed at 1-GHz, intermediate in those exposed at 1- and 10-GHz combined, and greatest in the 10-GHz group (most differences statistically significant). At all sites (with the exception of right subcutaneous), temperature values in the 1- and 10-GHz combined group were between those of the single-frequency exposure groups in both E and H orientations. During irradiation, arterial blood pressure initially increased and then decreased until death. Heart rate increased throughout the exposure period. The general, overall patterns of these changes were similar in all groups. The results indicate that no unusual physiological responses occur during multi-frequency MW exposure, when compared with results of single-frequency exposure. Bioelectromagnetics 21:159-166, 2000. Published 2000 Wiley-Liss, Inc.

The effect of ionizing irradiation on type I collagen of the tail in growing mice: a histology and electron microscopy study.

In order to examine the effect of radiation on growing tissue, especially the fibroblasts and their end-product, the collagen fibres, tails from 24 mice were irradiated at an age of 8 days with 20 Gy and 30 Gy (60Co). Tails from 18 animals served as controls. Six mice from each group were sacrificed on day 8, 20 and 30. Transmission electron microscopy was used to examine the fibroblasts and the collagen fibrils. Non-irradiated fibroblasts had a nucleus rich in chromatin and an abundant endoplasmic reticulum with cisternae and condensing vacuoles. On day 20, approximately 50%, and on day 30, 25% of the fibroblasts irradiated with 30 Gy had a sparse endoplasmic reticulum pointing to a reduction of protein synthesis. While, on day 20, the fibrils irradiated with 20 Gy and with 30 Gy had significantly larger diameters compared to the controls, on day 30, the irradiated fibrils had a notably smaller diameter compared to the controls; 30 Gy-fibrils were larger than the 20 Gy-fibrils on both days. On day 20, the binding mean value of the 30 Gy-fibrils exceeded that of the controls and was significantly higher than that of the 20 Gy-fibrils, which was lower, though not significantly, than the controls. On day 30, the banding mean value of the 30 Gy-fibrils was notably lower than the control; and the value of the 20 Gy-fibrils was significantly lower than that of the 30 Gy-fibrils. The results are explained as an edema together with an inhibitory effect on the protein synthesis of the fibroblasts caused by the irradiation. This deduction is further supported by light microscopy of the tails.

Dynamic capillaroscopy: a minimally invasive technique for assessing photodynamic effects in vivo.

A noninvasive method for visualizing the microvasculature in the mouse tail is described, consisting of a custom-built microscope with through-lens illumination. The microscope is fitted with a television camera and images can be recorded on videotape and displayed on a television monitor. Blood vessels are imaged as columns of red blood cells, in which flow is clearly observed. Administration of photosensitizers and illumination with the standard light source produces no observable photodynamic effect on blood flow. The combination of photosensitizer and a more intense light source (either broadband light from a filtered mercury arc or red light from a laser) causes photodynamic cessation of flow within a few minutes. The magnitude of the effect is dependent on the dose and nature of the photosensitizer, the delay after photosensitization and the match between the laser light and the absorption spectra of the photosensitizers in the red region. We conclude that the technique yields results consistent with the known photodynamic effects of the photosensitizers in tumors and propose its use as an initial screening method in vivo, as a means of conducting pharmacokinetic experiments and as an assay of prolonged cutaneous photosensitivity.

Studies relevant to a means of quantifying the effects of hyperthermia.

There is as yet no fully satisfactory method of defining 'thermal dose'. However, in order to relate different hyperthermal treatments it has been suggested that the relationship between heating time and temperature be used to derive an isoeffect parameter which might be useful in this context. The equation t2/t1 = RT1-T2, where t is the treatment time, T the temperature, R = 2 for T greater than 42.5 degrees C and R = approximately 6 for T less than 42.5 degrees C, has been shown to fit the isoeffect data from many studies both in vivo and in vitro. Whether or not this relationship is applicable when the temperature is varying has been tested using an animal model system, i.e. the response of the baby rat tail. It has shown that the relationship between time and temperature is indeed characterized by the above equation, and the phenomena of thermotolerance and step-down sensitization were clearly demonstrated. Tails were then alternated between waterbaths at different temperatures in order to simulate varying temperature. The measured effects were, in general, in good agreement with those derived from the formula. The maximum difference between the observed and predicted effect, obtained under rather extreme conditions unlikely to be experienced clinically, amounted to an under-estimate of the effective temperature by approximately 0.3 degrees C, i.e. a net small effect of thermotolerance. It is therefore suggested that the above formula for 'heat dose' provides a reasonable interim method for comparing clinical hyperthermic treatments. The formula does not, however, account for differing absolute sensitivities between tissues nor for effects of fractionating heat treatments.

Lack of differential sparing of late ischaemic atrophy and early epidermal healing, after dose fractionation of mouse tails down to 2.6 Gy per fraction.

Long-term atrophy of irradiated mouse tails began after about 5 months, and the incidence rose steadily to the end of the lifespan. The major associated histological change was atherosclerosis in the single tail artery. The incidence of the ischaemic atrophy was dependent on the size of the irradiated volume. The probability of ischaemic atrophy assessed at 3 years after irradiation was little dependent on the dose. The fractionation effect was described by alpha/beta congruent to 30 Gy, which was not lower than the range of values applicable for healing of the early epidermal reactions on the tail. Hence the general finding of a sparing of late effects in tissues using low doses per fraction was not observed in these experiments using dose fractions down to 2.6 Gy and the present endpoints.