An experimental setup for proton irradiation of a murine leg model for radiobiological studies.

BACKGROUND: The purpose of this study was to introduce an experimental radiobiological setup used for in vivo irradiation of a mouse leg target in multiple positions along a proton beam path to investigate normal tissue- and tumor models with varying linear energy transfer (LET). We describe the dosimetric characterizations and an acute- and late-effect assay for normal tissue damage. METHODS: The experimental setup consists of a water phantom that allows the right hind leg of three to five mice to be irradiated at the same time. Absolute dosimetry using a thimble (Semiflex) and a plane parallel (Advanced Markus) ionization chamber and Monte Carlo simulations using Geant4 and SHIELD-HIT12A were applied for dosimetric validation of positioning along the spread-out Bragg peak (SOBP) and at the distal edge and dose fall-off. The mice were irradiated in the center of the SOBP delivered by a pencil beam scanning system. The SOBP was 2.8 cm wide, centered at 6.9 cm depth, with planned physical single doses from 22 to 46 Gy. The biological endpoint was acute skin damage and radiation-induced late damage (RILD) assessed in the mouse leg. RESULTS: The dose-response curves illustrate the percentage of mice exhibiting acute skin damage, and at a later point, RILD as a function of physical doses (Gy). Each dose-response curve represents a specific severity score of each assay, demonstrating a higher ED50 (50% responders) as the score increases. Moreover, the results reveal the reversible nature of acute skin damage as a function of time and the irreversible nature of RILD as time progresses. CONCLUSIONS: We want to encourage researchers to report all experimental details of their radiobiological setups, including experimental protocols and model descriptions, to facilitate transparency and reproducibility. Based on this study, more experiments are being performed to explore all possibilities this radiobiological experimental setup permits.

Rat mesenteric small artery neurogenic dilatation is predominantly mediated by ß1 -adrenoceptors in vivo.

KEY POINTS: The prevailing dogma about neurogenic regulation of vascular tone consists of major vasodilatation caused by CGRP (and possibly substance P) released from sensory-motor nerves and vasoconstriction caused by noradrenaline, ATP and neuropeptode Y release from sympathetic nerves. Most studies on perivascular nerve-mediated vasodilatation are made in vitro. In the present study, we provide evidence indicating that in vivo electrical perivascular nerve stimulation in rat mesenteric small arteries causes a large ß1-adrenoceptor-mediated vasodilatation, which contrasts with a smaller vasodilatation caused by endogenous CGRP that is only visible after inhibition of Y1 NPY receptors. ABSTRACT: Mesenteric arteries are densely innervated and the nerves are important regulators of vascular tone and hence blood pressure and blood flow. Perivascular sensory-motor nerves have been shown to cause vasodilatation in vitro. However, less is known about their function in vivo. Male Wistar rats (10-12 weeks old; n = 72) were anaesthetized with ketamine (3 mg kg-1 ) and xylazine (0.75 mg kg-1 ) or pentobarbital (60 mg kg-1 ). After a laparotomy, a section of second-order mesenteric artery was visualized in an organ bath after minimal removal of perivascular adipose tissue. The effects of electrical field stimulation (EFS) and drugs on artery diameter and blood flow were recorded with intravital microscopy and laser speckle imaging. EFS caused vasodilatation in arteries constricted with 1 µm U46619 in the presence of 140 µm suramin and 1 µm prazosin. The vasodilatation was inhibited by 1 µm tetrodotoxin and 5 µm guanethidine, although not by the 1 µm of the CGRP receptor antagonist BIBN4096bs. In the presence of 0.3 µm Y1 receptor antagonist BIBP3226, BIBN4096bs partly inhibited the vasodilatation. Atenolol at a concentration 1 µm inhibited the vasodilatation, whereas 0.1 µm of the ß2 -adrenoceptor selective antagonist ICI-118,551 had no effect. Increasing the extracellular [K+ ] to 20 mm caused vasodilatation but was converted to vasoconstriction in the presence of 1 µm BIBN4096bs, and constriction to 30 mm potassium was potentiated by BIBN4096bs. Atenolol but not BIBN4096bs increased contraction to EFS in the absence of suramin and prazosin. In mesenteric small arteries of anaesthetized rats, EFS failed to stimulate major dilatation via sensory-motor nerves but induced sympathetic ß1 -adrenoceptor-mediated dilatation.

Allosteric mechanisms underlying the adaptive increase in hemoglobin-oxygen affinity of the bar-headed goose.

The high blood-O2 affinity of the bar-headed goose (Anser indicus) is an integral component of the biochemical and physiological adaptations that allow this hypoxia-tolerant species to undertake migratory flights over the Himalayas. The high blood-O2 affinity of this species was originally attributed to a single amino acid substitution of the major hemoglobin (Hb) isoform, HbA, which was thought to destabilize the low-affinity T state, thereby shifting the T-R allosteric equilibrium towards the high-affinity R state. Surprisingly, this mechanistic hypothesis has never been addressed using native proteins purified from blood. Here, we report a detailed analysis of O2 equilibria and kinetics of native major HbA and minor HbD isoforms from bar-headed goose and greylag goose (Anser anser), a strictly lowland species, to identify and characterize the mechanistic basis for the adaptive change in Hb function. We find that HbA and HbD of bar-headed goose have consistently higher O2 affinities than those of the greylag goose. The corresponding Hb isoforms of the two species are equally responsive to physiological allosteric cofactors and have similar Bohr effects. Thermodynamic analyses of O2 equilibrium curves according to the two-state Monod-Wyman-Changeaux model revealed higher R-state O2 affinities in the bar-headed goose Hbs, associated with lower O2 dissociation rates, compared with the greylag goose. Conversely, the T state was not destabilized and the T-R allosteric equilibrium was unaltered in bar-headed goose Hbs. The physiological implication of these results is that increased R-state affinity allows for enhanced O2 saturation in the lungs during hypoxia, but without impairing O2 delivery to tissues.

In vivo validation and tissue sparing factor for acute damage of pencil beam scanning proton FLASH.

BACKGROUND AND PURPOSE: Preclinical studies indicate a normal tissue sparing effect using ultra-high dose rate (FLASH) radiation with comparable tumor response. Most data so far are based on electron beams with limited utility for human treatments. This study validates the effect of proton FLASH delivered with pencil beam scanning (PBS) in a mouse leg model of acute skin damage and quantifies the normal tissue sparing factor, the FLASH factor, through full dose response curves. MATERIALS AND METHODS: The right hind limb of CDF1 mice was irradiated with a single fraction of proton PBS in the entrance plateau of either a 244 MeV conventional dose rate field or a 250 MeV FLASH field. In total, 301 mice were irradiated in four separate experiments, with 7-21 mice per dose point. The endpoints were the level of acute moist desquamation to the skin of the foot within 25 days post irradiation. RESULTS: The field duration and field dose rate were 61-107 s and 0.35-0.40 Gy/s for conventional dose rate and 0.35-0.73 s and 65-92 Gy/s for FLASH. Full dose response curves for five levels of acute skin damage for both conventional and FLASH dose rate revealed a distinct normal tissue sparing effect with FLASH: across all scoring levels, a 44-58% higher dose was required to give the same biological response with FLASH as compared to the conventional dose rate. CONCLUSIONS: The normal tissue sparing effect of PBS proton FLASH was validated. The FLASH factor was quantified through full dose response curves.